Method of treating type I diabetes using an AAV vector encoding uracortin 2

ABSTRACT

In alternative embodiments, the invention provides methods for treating, ameliorating or protecting (preventing) an individual or a patient against a disease, an infection or a condition responsive to an increased paracrine polypeptide level in vivo comprising: providing a paracrine polypeptide-encoding nucleic acid or gene operatively linked to a transcriptional regulatory sequence; or an expression vehicle, a vector, a recombinant virus, or equivalent, having contained therein a paracrine-encoding nucleic acid or gene, and the expression vehicle, vector, recombinant virus, or equivalent can express the paracrine-encoding nucleic acid or gene in a cell or in vivo; and administering or delivering the paracrine polypeptide-encoding nucleic acid or gene operatively linked to a transcriptional regulatory sequence, or the expression vehicle, vector, recombinant virus, or equivalent, to an individual or a patient in need thereof, thereby treating, ameliorating or protecting (preventing) the individual or patient against the disease, infection or condition responsive to an increased paracrine polypeptide level.

CROSS-REFERENCE TO RELATED APPLICATIONS

This United States utility patent application is a continuation of U.S.Ser. No. 15/625,719, filed Jun. 16, 2017, now U.S. Pat. No. 10,202,618,which is a continuation-in-part (CIP) of U.S. Ser. No. 14/378,645, filedAug. 13, 2014 (now abandoned), which is a § 371 national phase of PCTinternational patent application no. PCT/US2013/025997, having aninternational filing date of Feb. 13, 2013, which claims benefit ofpriority to U.S. Provisional Patent Application Ser. No. 61/598,772,filed Feb. 14, 2012. The aforementioned applications are expresslyincorporated herein by reference in their entirety and for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with government support under grant No. HL088426awarded by the National Institutes of Health (NIH), DHHS. The governmenthas certain rights in the invention.

TECHNICAL FIELD

This invention relates to cellular and molecular biology and medicine.The invention provides compositions and in vitro and ex vivo methods. Inalternative embodiments, the invention provides methods for treating,ameliorating or protecting (preventing) an individual or a patientagainst a disease, an infection or a condition responsive to anincreased or a sustained paracrine polypeptide level in vivo comprising:providing a paracrine polypeptide-encoding nucleic acid or geneoperatively linked to a transcriptional regulatory sequence; or anexpression vehicle, a vector, a recombinant virus, or equivalent, havingcontained therein a paracrine-encoding nucleic acid, gene, transcript ormessage, and the expression vehicle, vector, recombinant virus, orequivalent can express the paracrine-encoding encoding nucleic acid,gene, transcript or message in a cell or in vivo; and administering ordelivering the paracrine polypeptide-encoding nucleic acid, gene,transcript or message operatively linked to a transcriptional regulatorysequence, or the expression vehicle, vector, recombinant virus, orequivalent, to an individual or a patient in need thereof, therebytreating, ameliorating or protecting (preventing) the individual orpatient against the disease, infection or condition responsive to anincreased paracrine polypeptide level.

BACKGROUND

Recently an intravenous injection of a virus vector encoding humanFactor IX, which is deficient in Hemophilia B was shown to increaseFactor IX concentration in the serum of subjects with Hemophilia B to adegree that lowered their requirements for exogenous Factor IX infusion.However: 1) this protein was not under regulated expression, andtherefore, did not enable optimal tailoring of levels of the transgenein the serum, 2) this system did not provide for a means to turn offtransgene expression in case of undesired or unexpected effects, and 3)the gene, Factor IX, was not a paracrine gene, and had no beneficialcardiovascular effects, and therefore, could not be used to treat heartdisease.

SUMMARY

The invention provides methods for treating, ameliorating or protecting(preventing) an individual or a patient against any disease, infectionor condition responsive to an increased paracrine polypeptide level invivo. In alternative embodiments, the invention provides methods fortreating, ameliorating or protecting (preventing) against a disease, aninfection or a condition responsive to an increased or sustained peptideor paracrine polypeptide level in vivo comprising:

(a) (i) providing a paracrine polypeptide-encoding nucleic acid or geneoperatively linked to a transcriptional regulatory sequence; or anexpression vehicle, a vector, a recombinant virus, or equivalent, havingcontained therein a paracrine-encoding nucleic acid or gene, or aparacrine polypeptide-expressing nucleic acid, transcript or message,and the expression vehicle, vector, recombinant virus, or equivalent canexpress the paracrine-encoding nucleic acid, gene, transcript or messagein a cell or in vivo; and

(ii) administering or delivering the paracrine polypeptide-encodingnucleic acid, gene, transcript or message operatively linked to atranscriptional regulatory sequence, or the expression vehicle, vector,recombinant virus, or equivalent, to the cell, or an individual or apatient in need thereof,

thereby treating, ameliorating or protecting (preventing) the individualor patient against the disease, infection or condition responsive to anincreased or a sustained paracrine polypeptide level;

(b) the method of (a), wherein the expression vehicle, vector,recombinant virus, or equivalent is or comprises:

an adeno-associated virus (AAV), a lentiviral vector or an adenovirusvector,

an AAV serotype AAV5, AAV6, AAV8 or AAV9,

a rhesus-derived AAV, or the rhesus-derived AAV AAVrh.10hCLN2,

an AAV capsid mutant or AAV hybrid serotype,

an organ-tropic AAV, or a cardiotropic AAV, or a cardiotropic AAVM41mutant,

wherein optionally the AAV is engineered to increase efficiency intargeting a specific cell type that is non-permissive to a wild type(wt) AAV and/or to improve efficacy in infecting only a cell type ofinterest,

and optionally the hybrid AAV is retargeted or engineered as a hybridserotype by one or more modifications comprising: 1) a transcapsidation,2) adsorption of a bi-specific antibody to a capsid surface, 3)engineering a mosaic capsid, and/or 4) engineering a chimeric capsid;

(c) the method of (a), wherein the paracrine-encoding nucleic acid,gene, transcript or message is operatively linked to a regulated orinducible transcriptional regulatory sequence;

(d) the method of (c), wherein the regulated or inducibletranscriptional regulatory sequence is a regulated or induciblepromoter,

wherein optionally a positive (an activator) and/or a negative (arepressor) modulator of transcription and/or translation is operablylinked to the paracrine polypeptide-encoding nucleic acid, gene,transcript or message;

(e) the method of any of (a) to (d), wherein administering the paracrinepolypeptide-encoding nucleic acid, gene, transcript or messageoperatively linked to a transcriptional regulatory sequence, or theexpression vehicle, vector, recombinant virus, or equivalent, to anindividual or a patient in need thereof results in a paracrine proteinbeing released into the bloodstream or general circulation, or anincreased or sustained expression of the paracrine protein in the cell,

wherein optionally the release or increased or sustained expression ofthe paracrine protein is dependent on activation of an induciblepromoter, or de-repression of a repressor, operably linked to theparacrine polypeptide-encoding nucleic acid, gene, transcript ormessage; or

(f) the method of any of (a) to (e), wherein the disease, infection orcondition responsive to an increased paracrine polypeptide level in vivois a cardiac contractile dysfunction; a congestive heart failure (CHF);a cardiac fibrosis; a cardiac myocyte disease, dysfunction or apoptosis;a pulmonary hypertension; a heart, skin, liver, lung, muscle, nerve,brain or kidney disease, cancer or dysfunction; a cancer or a neoplasia;or, a hemophilia or a Hemophilia B.

In alternative embodiments of methods of the invention:

(a) the paracrine-encoding nucleic acid or gene operatively linked tothe transcriptional regulatory sequence; or the expression vehicle,vector, recombinant virus, or equivalent, is administered or deliveredto the individual or a patient in need thereof, by oral, intramuscular(IM) injection, by intravenous (IV) injection, by subcutaneous (SC) orintradermal injection, by intrathecal injection, by intra-arterial (IA)injection, by intracoronary injection, by inhalation, or by a biolisticparticle delivery system, or by using a “gene gun”, air pistol or aHELIOS™ gene gun (Bio-Rad Laboratories, Hercules, Calif.); or

(b) the paracrine-encoding nucleic acid or gene operatively linked tothe transcriptional regulatory sequence; or the expression vehicle,vector, recombinant virus, or equivalent, is administered or deliveredto the individual or a patient in need thereof, by introduction into anytissue or fluid space within the body that is adjacent to or is drainedby the bloodstream, such that the encoded protein may be secreted fromcells in the tissue and released into the bloodstream.

In alternative embodiments of methods of the invention: the paracrinepolypeptide or peptide is or comprises: a mammalian cardiotonic peptide,a growth factor, a Serelaxin, a Relaxin-2, a Urocortin-2 (UCn-2), aUrocortin-1 (UCn-1), a Urocortin-3 (UCn-3), a Brain Natriuretic Peptide,a Prostacyclin Synthase, a Growth Hormone, an Insulin-like GrowthFactor-1, or any combination thereof; or, a human cardiotonic peptide, ahuman growth factor, a Serelaxin, a Relaxin-2, a Urocortin-2, aUrocortin-1, a Urocortin-3, a Brain Natriuretic Peptide, a ProstacyclinSynthase, a Growth Hormone, an Insulin-like Growth Factor-11, or anycombination thereof.

In alternative embodiments of methods of the invention: the paracrinepolypeptide is a Urocortin, a Urocortin-2, a Urocortin-1, a Urocortin-3,a Relaxin-2 or a Brain Natriuretic Peptide and the disease or conditionis a congestive heart failure (CHF); or the paracrine polypeptide isProstacyclin Synthase and the disease or condition a pulmonaryhypertension and the disease or condition is a congestive heart failure(CHF); or the paracrine polypeptide is Prostacyclin Synthase and thedisease or condition a pulmonary hypertension.

In alternative embodiments of methods of the invention:

(a) the individual, patient or subject is administered a stimulus orsignal that induces expression of the paracrine-expressing nucleic acidor gene, or induces or activates a promoter (e.g., operably linked tothe paracrine-expressing nucleic acid or gene) that induces expressionof the paracrine-expressing nucleic acid or gene;

(b) the individual, patient or subject is administered a stimulus orsignal that induces synthesis of an activator of a promoter, optionallya paracrine-expressing nucleic acid or gene-specific promoter (e.g.,operably linked to the paracrine-expressing nucleic acid or gene);

(c) the individual, patient or subject is administered a stimulus orsignal that induces synthesis of a natural or a synthetic activator ofthe paracrine-expressing nucleic acid or gene or theparacrine-expressing nucleic acid or gene-specific promoter,

wherein optionally the natural activator is an endogenous transcriptionfactor;

(d) the method of (c), wherein the synthetic activator is a zinc-fingerDNA binding protein designed to specifically and selectively turn on anendogenous or exogenous target gene, wherein optionally the endogenoustarget is a gene paracrine-expressing nucleic acid or gene or anactivator of a paracrine-expressing nucleic acid or gene, or anactivator of a promoter operatively linked to a paracrine-expressingnucleic acid or gene;

(e) the method of any of (a) to (c), wherein the stimulus or signalcomprises a biologic, a light, a chemical or a pharmaceutical stimulusor signal;

(f) the individual, patient or subject is administered a stimulus orsignal that stimulates or induces expression of a post-transcriptionalactivator of a paracrine-expressing nucleic acid or gene, or anactivator of a promoter operatively linked to a paracrine-expressingnucleic acid or gene, or

(g) the individual, patient or subject is administered a stimulus orsignal that inhibits or induces inhibition of a transcriptionalrepressor or a post-transcriptional repressor of a paracrine-expressingnucleic acid or gene.

In alternative embodiments of methods of the invention: the chemical orpharmaceutical that induces expression of the paracrine-expressingnucleic acid or gene, or induces expression of the regulated orinducible promoter operatively linked to the paracrine-expressingnucleic acid or gene, is an oral antibiotic, a doxycycline or arapamycin; or a tet-regulation system using doxycycline is used toinduce expression of the paracrine-expressing nucleic acid or gene, oran equivalent thereof.

In alternative embodiments of methods of the invention: theparacrine-expressing nucleic acid or gene or the expression vehicle,vector, recombinant virus, or equivalent, is formulated in a liquid, agel, a hydrogel, a powder or an aqueous formulation.

In alternative embodiments of methods of the invention: theparacrine-expressing nucleic acid or gene or the expression vehicle,vector, recombinant virus, or equivalent, or the urocortin-2 (UCn-2)peptide or polypeptide, is formulated in a vesicle, liposome,nanoparticle or nanolipid particle (NLP) or equivalents, or formulatedfor delivery using a vesicle, liposome, nanoparticle or nanolipidparticle (NLP) or equivalents.

In alternative embodiments of methods of the invention: theparacrine-expressing nucleic acid or gene or the expression vehicle,vector, recombinant virus, or equivalent, is formulated in, or insertedor transfected into, an isolated or cultured cell, and optionally thecell is a mammalian cell, a cardiac cell, or a human cell, a non-humanprimate cell, a monkey cell, a mouse cell, a rat cell, a guinea pigcell, a rabbit cell, a hamster cell, a goat cell, a bovine cell, anequine cell, an ovine cell, a canine cell or a feline cell.

In alternative embodiments of methods of the invention: theparacrine-expressing nucleic acid or gene or the expression vehicle,vector, recombinant virus, or equivalent, or the urocortin-2 (UCn-2)peptide or polypeptide, is formulated as a pharmaceutical or a sterileformulation.

In alternative embodiments of methods of the invention: theparacrine-expressing nucleic acid or gene or the expression vehicle,vector, recombinant virus, or equivalent, or the urocortin-2 (UCn-2)peptide or polypeptide, is formulated or delivered with, on, or inconjunction with a product of manufacture, an artificial organ or animplant.

In alternative embodiments of methods of the invention: theparacrine-expressing nucleic acid or gene or the expression vehicle,vector, recombinant virus, or equivalent expresses a paracrinepolypeptide in vitro or ex vivo.

In alternative embodiments the invention provides methods for treating,ameliorating or protecting (preventing) an individual or a patientagainst a paracrine-responsive pathology, infection, disease, illness,or condition, comprising practicing a method of the invention.

In alternative embodiments the invention provides methods for treating,ameliorating or protecting (preventing) a cardiac contractiledysfunction; a congestive heart failure (CHF); a cardiac fibrosis; acardiac myocyte disease, dysfunction or apoptosis; a pulmonaryhypertension; a heart, skin, liver, lung, muscle, nerve, brain or kidneydisease, cancer or dysfunction; a cancer or a neoplasia; or, ahemophilia or a Hemophilia B, comprising practicing a method of theinvention.

In alternative embodiments, the invention provides methods of treating,ameliorating or protecting (preventing) diabetes or pre-diabetes in apatient or an individual comprising:

(a) practicing a method of the invention, wherein the paracrinepolypeptide or peptide comprises or consists of a urocortin-2 (UCn-2);and

(b) administering a urocortin-2 (UCn-2) peptide or polypeptide, or anucleic acid, gene, message or transcript encoding a urocortin-2 (UCn-2)to an individual or patient in need thereof,

wherein optionally the urocortin-2 (UCn-2) peptide or polypeptide is anisolated, a recombinant, a synthetic and/or a peptidomimetic peptide orpolypeptide or variant thereof,

thereby treating, ameliorating or protecting (preventing) the diabetesor pre-diabetes in the patient or individual.

In alternative embodiments, the invention provides methods of treating,ameliorating or protecting (preventing) obesity in a patient or anindividual comprising:

(a) practicing a method of the invention, wherein the paracrinepolypeptide or peptide comprises or consists of a urocortin-2 (UCn-2);and

(b) administering a urocortin-2 (UCn-2) peptide or polypeptide, or anucleic acid, gene, message or transcript encoding a urocortin-2 (UCn-2)to an individual or patient in need thereof,

wherein optionally the urocortin-2 (UCn-2) peptide or polypeptide is anisolated, a recombinant, a synthetic and/or a peptidomimetic peptide orpolypeptide or variant thereof,

thereby treating, ameliorating or protecting (preventing) the obesity inthe patient or individual.

In alternative embodiments, the invention provides methods ofsuppressing weight gain, or suppressing the appetite, or stimulating orinitiating weight loss, in a patient or an individual comprising:

(a) practicing a method of the invention, wherein the paracrinepolypeptide or peptide comprises or consists of a urocortin-2 (UCn-2);and

(b) administering a urocortin-2 (UCn-2) peptide or polypeptide, or anucleic acid, gene, message or transcript encoding a urocortin-2 (UCn-2)to an individual or patient in need thereof,

wherein optionally the urocortin-2 (UCn-2) peptide or polypeptide is anisolated, a recombinant, a synthetic and/or a peptidomimetic peptide orpolypeptide or variant thereof,

thereby suppressing weight gain, or suppressing the appetite, orstimulating or initiating weight loss, in the patient or individual.

In alternative embodiments, the urocortin-2 (UCn-2) peptide orpolypeptide is formulated in or as a vesicle, liposome, nanoparticle ornanolipid particle (NLP), or is formulated for: oral administration,intramuscular (IM) injection, intravenous (IV) injection, subcutaneous(SC) or intradermal injection, intrathecal injection, intra-arterial(IA) injection, intracoronary injection, inhalation, or administrationby aerosol.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications cited herein are herebyexpressly incorporated by reference for all purposes.

DESCRIPTION OF DRAWINGS

FIG. 1 illustrates an exemplary construct of the invention comprisingAAV5 encoding IGF1, as described in Example 2, below.

FIGS. 2A-B illustrate data from studies where cultured neonatal ratcardiac myocytes were infected with the exemplary AAV5.IGFI.tetconstruct of the invention, and IGFI was induced, expressed, and thenmeasured, as described in Example 2, below.

FIG. 3 graphically illustrates regulated expression of IGFI mRNAexpression in cultured neonatal rat cardiac myocytes after gene transferwith the exemplary AAV5.IGFI-tet adding, and them removing, doxicillin,as described in Example 2, below.

FIGS. 4A-B illustrates photomicrographs showing EGFP expression inunilateral tibialis anterior muscle 3 weeks after AAV5.EGFP genetransfer in rats; and FIG. 4B is Table 4, which summarizes data from theechocardiography measuring the effects of Skeletal Muscle IGFIExpression in CHF, as described in Example 2, below.

FIG. 5 illustrates the experimental protocol for gene transfer of theexemplary AAV5.IGFI.tet of the invention in skeletal muscle in CHF, asdescribed in Example 2, below.

FIGS. 6A-C illustrates the effects of AAV5.IGFI-tet gene transfer oncardiac apoptosis and fibrosis: FIG. 6A graphically illustrates datafrom TUNEL staining that indicated that activation of IGFI expression(IGF-On) was associated with reduced cardiac myocyte apoptosis; FIG. 6Billustrates picrosirius red-stained sections of the uninfarctedintraventricular septum from IGF-Off and IGF-On rats that showed reducedcardiac fibrosis, and collagen fractional area was reduced; and FIG. 6Cgraphically illustrates this data from the IGF-Off and IGF-On rats, asdescribed in Example 2, below.

FIG. 7 graphically illustrates that intravenous gave better results thanintramuscular administration in increasing serum levels of IGFI when anexemplary when AAV5 construct of the invention was administered:intravenous delivery in mice, intramuscular delivery in rats, asdescribed in Example 2, below.

FIG. 8 graphically, and by image, illustrates data showing the relativeefficacy of intravenous delivery of exemplary AAV5 and AAV9 constructsof the invention using copy number and transgene expression in liver andheart as endpoints, as described in Example 2, below.

FIG. 9 illustrates an exemplary protocol for determining and testing themost appropriate vector to use for a desired or a particular indicationwhen practicing a method of the invention, as discussed in Example 2,below.

FIGS. 10A-F illustrate exemplary vector constructs of the invention, asdescribed in Example 2, below.

FIG. 11 graphically illustrates data showing that IV AAV8 is the optimalvector and delivery route to attain sustained increased levels of serumurocortin-2 (UCn-2) for a paracrine approach, as described in Example 3,below.

FIGS. 12A-B graphically illustrates a time course of UCn2 mRNAexpression in liver after IV administration of the exemplaryAAV8.CBA.UCn2 construct; and FIG. 12B graphically illustrates datashowing UCn2 mRNA expression in LV 6 weeks after AAV8.CBA.UCn2 IVadministration, as described in Example 3, below.

FIGS. 13A-B graphically illustrates data from a study to determine ifUCn2 gene transfer increased LV function by delivery of the exemplaryAAV8.UCn2 construct of the invention by intravenous (IV) delivery innormal mice: FIG. 13A graphically illustrates data showing UCn2 genetransfer increased LV contractile function; FIG. 13B graphicallyillustrates data showing −dP/dt also was reduced, indicating enhanced LVrelaxation, as described in Example 3, below.

FIGS. 14A-C illustrates data showing the effects of UCn2 transfer on thefailing heart: FIG. 14A illustrates the study protocol; and FIGS. 14Band 14C illustrate data showing the effects of UCn2 transfer on thefailing heart, as described in Example 3, below.

FIGS. 15A-B illustrates data, FIG. 15A by graph, FIG. 15B by immunoblot,where normal mice received IV delivery of the exemplary AAV8.CBA.UCn2;and four weeks later, LV samples from the UCn2 gene transfer groupshowed a 2-fold increase in SERCA2a protein expression, as described inExample 3, below.

FIGS. 16A-B shows data of Ca²⁺ transients following UCn2 gene transfer:FIG. 16A graphically illustrates that UCn2 gene transfer increased therate of Ca²⁺ decline; FIG. 16B graphically illustrates that time-to-Ca²⁺transient decay was shortened in cardiac myocytes from mice that hadreceived UCN2 gene transfer 4 w prior, as described in Example 3, below.

FIGS. 17A-B shows data that UCn2 protects cultured neonatal rat cardiacmyocytes from hypoxic injury: FIG. 17A illustrates that UCn2 preservesmorphological normality 24 hr after NaN₃ treatment; FIG. 17B graphicallyillustrates that UCn2 reduced LDH release after NaN₃ treatment, asdescribed in Example 3, below.

FIGS. 18A-B graphically illustrates that phosphorylation of both CREB(FIG. 18A) and β-catenin (FIG. 18B) was detected in LV samples 4 w afterIV delivery of the exemplary UCn2.CBA.UCn2 construct of the invention,as described in Example 3, below.

FIGS. 19A-B illustrates data showing UCn2 affects glucose regulation:Mice received IV delivery of the exemplary AAV8.CBA.UCn2: FIG. 19Aillustrates that a small reduction in fasting blood glucose was seen inthe UCn2 group: FIG. 19B illustrates results indicating that UCn2 genetransfer promotes glucose utilization and protects against diet-inducedhyperglycemia, as described in Example 3, below.

FIGS. 20A-F illustrate exemplary constructs of the invention, asdescribed in Example 3, below.

FIG. 21 shows reduced fasting glucose (p<0.0001) in mice that receivedAAV8.UCn2.

FIG. 22 shows hemoglobin A1C (p=0.002) in mice that received AAV8.UCn2.

FIG. 23 shows a glucose tolerance test (p=0.01) in mice that receivedAAV8.UCn2.

FIG. 24 shows Insulin Release in mice that received AAV8.UCn2 (p=0.003at 60 min and p=0.004 at 120 min).

FIG. 25 shows Insulin Tolerance in mice that received AAV8.UCn2(p<0.003).

FIG. 26 illustrates Table 5.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The invention provides compositions and in vivo and ex vivo methodscomprising administration of paracrine-encoding nucleic acids, genes,transcripts or messages to treat, ameliorate or protect (as aprophylaxis) individuals against diseases, infections or conditionsresponsive to increased paracrine levels in vivo. In alternativeembodiments, the invention provides compositions and methods for the invivo or in situ delivery and/or in vivo expression of, and controlledexpression of, any paracrine polypeptide or peptide, e.g., a mammaliancardiotonic peptide, a Serelaxin, a Relaxin-2, a Urocortin-2, aUrocortin-1, a Urocortin-3, a Brain Natriuretic Peptide, a ProstacyclinSynthase, a Growth Hormone, an Insulin-like Growth Factor-1, or anycombination thereof; or, a human cardiotonic peptide, a Serelaxin, aRelaxin-2, a Urocortin-2, a Urocortin-1, a Urocortin-3, a BrainNatriuretic Peptide, a Prostacyclin Synthase, a Growth Hormone, anInsulin-like Growth Factor-1, or any combination thereof.

In alternative embodiments, the invention provides compositions andmethods for the delivery and controlled expression of aparacrine-encoding nucleic acid or gene, or an expression vehicle (e.g.,vector, recombinant virus, and the like) comprising (having containedtherein) a paracrine-encoding nucleic acid or gene, that results in aparacrine protein being released into the bloodstream or generalcirculation where it can have a beneficial effect on in the body, e.g.,such as the heart in the case of treating cardiovascular disease, or thelungs or kidneys, or other targets.

In alternative embodiments, the invention provides expression vehicles,vectors, recombinant viruses and the like for in vivo expression of aparacrine-encoding nucleic acid or gene to practice the methods of thisinvention. In alternative embodiments, the expression vehicles, vectors,recombinant viruses and the like expressing the paracrine-encodingnucleic acid or gene can be delivered by intramuscular (IM) injection,by intravenous (IV) injection, by subcutaneous injection, by inhalation,by a biolistic particle delivery system (e.g., a so-called “gene gun”),and the like, e.g., as an outpatient, e.g., during an office visit.

In alternative embodiments, this “peripheral” mode of delivery, e.g.,expression vehicles, vectors, recombinant viruses and the like injectedIM or IV, can circumvent problems encountered when genes or nucleicacids are expressed directly in an organ (e.g., the heart, lung orkidney) itself. Sustained secretion of a desired paracrine protein(s) inthe bloodstream or general circulation also circumvents the difficultiesand expense of administering proteins by infusion, which can beparticularly problematic for many proteins which exhibit very short halflives in the body, as summarized in Table 1, below:

TABLE 1 Peptide IV Infusion vs Gene Transfer Feature IV Infusion GeneTransfer Requires Hospitalization Most often No Indwelling CathetersOften No Infection Risk High No Thrombosis Risk High No Expense High LowEase of Use Low High “Mobility” of Therapy Low High Efficacy in CHF YesUntested Dosage Regulation Tight via Reg Expression “Mobility” refers toease of using when away from home (travelling, etc); Reg, Regulated (thepatient takes an oral agent in a dose that provides the desired level oftransgene expression)

In alternative embodiments, the invention provides methods for beingable to turn on and turn off paracrine-expressing nucleic acid or geneexpression easily and efficiently for tailored treatments and insuranceof optimal safety.

In alternative embodiments, the paracrine protein or proteins expressedby the paracrine-expressing nucleic acid(s) or gene(s) have a beneficialor favorable effects (e.g., therapeutic or prophylactic) on a tissue oran organ, e.g., the heart, blood vessels, lungs, kidneys, or othertargets, even though secreted into the blood or general circulation at adistance (e.g., anatomically remote) from their site or sites of action.

In an exemplary embodiment of the invention, a paracrine-expressingnucleic acid or gene encoding Urocortin-2 is used, but otherparacrine-expressing nucleic acids or genes can be used to practicemethods of this invention, including but not limited to, e.g., fortreating congestive heart failure (CHF) or pulmonary hypertension:Urocortin-1 and Urocortin-3, Brain Natriuretic Peptide (for CHF),Prostacyclin Synthase (for pulmonary hypertension), Growth Hormone,and/or Insulin-like Growth Factor-1, or any combination thereof.

In alternative embodiments the invention provides applications, andcompositions and methods, for a regulated expression system providingfor controlled expression of a paracrine-type gene to treat a heart orlung disease, e.g., congestive heart failure (CHF) or pulmonaryhypertension.

For example, in alternative embodiments a recombinant virus (e.g., along-term virus or viral vector), or a vector, or an expression vector,and the like, can be injected, e.g., in a systemic vein (e.g., IV), orby intramuscular (IM) injection, by inhalation, or by a biolisticparticle delivery system (e.g., a so-called “gene gun”), e.g., as anoutpatient, e.g., in a physician's office. In alternative embodiments,days or weeks later (e.g., four weeks later), the individual, patient orsubject is administered (e.g., inhales, is injected or swallows), achemical or pharmaceutical that induces expression of theparacrine-expressing nucleic acids or genes; for example, an oralantibiotic (e.g., doxycycline or rapamycin) is administered once daily(or more or less often), which will activate the expression of the gene.In alternative embodiments, after the “activation”, or inducement ofexpression (e.g., by an inducible promoter) of the nucleic acid or gene,a paracrine protein is synthesized and released into the subject'scirculation (e.g., into the blood), and subsequently has favorablephysiological effects, e.g., therapeutic or prophylactic, that benefitthe individual or patient (e.g., benefit heart, kidney or lungfunction), depending on the paracrine protein or proteins expressed.When the physician or subject desires discontinuation of the treatment,the subject simply stops taking the activating chemical orpharmaceutical, e.g., antibiotic.

The inventors have used an AAV vector encoding Urocortin-2 andadministered the vector to mice using intravenous delivery. The resultsshowed: 1) a 17-fold increase in serum levels of the transgene 4-6 weeksafter intravenous delivery of the vector; 2) pronounced favorableeffects on cardiac contractile function (systolic function); and 3)pronounced favorable effects on cardiac relaxation (diastolic function).

In alternative embodiments, applications of the present inventioninclude: the treatment of severe, low ejection fraction heart failure;the treatment of pulmonary hypertension; the treatment of heart failurewith preserved ejection fraction; replacement of current therapies thatrequire hospitalization and sustained intravenous infusions ofvasoactive peptides for the treatment of pulmonary hypertension andheart failure; and, the treatment of other conditions in whichcontrolled expression of a paracrine-type gene can be used to promotefavorable effects at a distance in the body.

Generating and Manipulating Nucleic Acids

In alternative embodiments, to practice the methods of the invention,the invention provides isolated, synthetic and/or recombinant nucleicacids or genes encoding paracrine polypeptides. In alternativeembodiments, to practice the methods of the invention, the inventionprovides paracrine-expressing nucleic acids or genes in recombinant formin an (e.g., spliced into) an expression vehicle for in vivo expression,e.g., in a vector or a recombinant virus. In other alternativeembodiments, the invention provides, e.g., isolated, synthetic and/orrecombinant nucleic acids encoding inhibitory nucleic acids (e.g.,siRNA, microRNA, antisense, ribozyme) that can inhibit the expression ofgenes or messages (mRNAs) that inhibit the expression of the desiredparacrine gene.

In alternative embodiments, nucleic acids of the invention are made,isolated and/or manipulated by, e.g., cloning and expression of cDNAlibraries, amplification of message or genomic DNA by PCR, and the like.The nucleic acids and genes used to practice this invention, includingDNA, RNA, iRNA, antisense nucleic acid, cDNA, genomic DNA, vectors,viruses or hybrids thereof, can be isolated from a variety of sources,genetically engineered, amplified, and/or expressed/generatedrecombinantly. Recombinant polypeptides (e.g., paracrine chimericproteins used to practice this invention) generated from these nucleicacids can be individually isolated or cloned and tested for a desiredactivity. Any recombinant expression system or gene therapy deliveryvehicle can be used, including e.g., viral (e.g., AAV constructs orhybrids) bacterial, fungal, mammalian, yeast, insect or plant cellexpression systems or expression vehicles.

Alternatively, nucleic acids used to practice this invention can besynthesized in vitro by well-known chemical synthesis techniques, asdescribed in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov(1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol.Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang(1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109;Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids used to practice thisinvention, such as, e.g., subcloning, labeling probes (e.g.,random-primer labeling using Klenow polymerase, nick translation,amplification), sequencing, hybridization and the like are welldescribed in the scientific and patent literature, see, e.g., Sambrook,ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, ColdSpring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULARBIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997);LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY:HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic AcidPreparation, Tijssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used topractice the methods of the invention is to clone from genomic samples,and, if desired, screen and re-clone inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in,e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

In alternative embodiments, to practice the methods of the invention,paracrine fusion proteins and nucleic acids encoding them are used. Anyparacrine polypeptide can be used to practice this invention, e.g., aUrocortin-1, a Urocortin-2, a Urocortin-3, a Brain Natriuretic Peptide,a Prostacyclin Synthase, a Growth Hormone, an Insulin-like GrowthFactor-1 protein. In alternative embodiments, the paracrine protein canbe fused to a heterologous peptide or polypeptide, such as a peptide fortargeting the polypeptide to a desired cell type, such a cardiacmyocytes, or a lung cell.

In alternative embodiments, a heterologous peptide or polypeptide joinedor fused to a protein used to practice this invention can be anN-terminal identification peptide which imparts a desiredcharacteristic, such as fluorescent detection, increased stabilityand/or simplified purification. Peptides and polypeptides used topractice this invention can also be synthesized and expressed as fusionproteins with one or more additional domains linked thereto for, e.g.,producing a more immunogenic peptide, to more readily isolate arecombinantly synthesized peptide, to identify and isolate antibodiesand antibody-expressing B cells, and the like. Detection andpurification facilitating domains include, e.g., metal chelatingpeptides such as polyhistidine tracts and histidine-tryptophan modulesthat allow purification on immobilized metals, protein A domains thatallow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of a cleavable linker sequences suchas Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between apurification domain and the motif-comprising peptide or polypeptide tofacilitate purification. For example, an expression vector can includean epitope-encoding nucleic acid sequence linked to six histidineresidues followed by a thioredoxin and an enterokinase cleavage site(see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998)Protein Expr. Purif. 12:404-414). The histidine residues facilitatedetection and purification while the enterokinase cleavage site providesa means for purifying the epitope from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

Nucleic acids or nucleic acid sequences used to practice this inventioncan be an oligonucleotide, nucleotide, polynucleotide, or to a fragmentof any of these, to DNA or RNA of genomic or synthetic origin which maybe single-stranded or double-stranded and may represent a sense orantisense strand, to peptide nucleic acid (PNA), or to any DNA-like orRNA-like material, natural or synthetic in origin. Compounds use topractice this invention include “nucleic acids” or “nucleic acidsequences” including oligonucleotide, nucleotide, polynucleotide, or anyfragment of any of these; and include DNA or RNA (e.g., mRNA, rRNA,tRNA, iRNA) of genomic or synthetic origin which may be single-strandedor double-stranded; and can be a sense or antisense strand, or a peptidenucleic acid (PNA), or any DNA-like or RNA-like material, natural orsynthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g.,e.g., double stranded iRNAs, e.g., iRNPs). Compounds use to practicethis invention include nucleic acids, i.e., oligonucleotides, containingknown analogues of natural nucleotides. Compounds use to practice thisinvention include nucleic-acid-like structures with synthetic backbones,see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197;Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996)Antisense Nucleic Acid Drug Dev 6:153-156. Compounds use to practicethis invention include “oligonucleotides” including a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandsthat may be chemically synthesized. Compounds use to practice thisinvention include synthetic oligonucleotides having no 5′ phosphate, andthus will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide can ligate to a fragment that has not beendephosphorylated.

In alternative aspects, compounds used to practice this inventioninclude genes or any segment of DNA involved in producing a paracrinepolypeptide (e.g., a Urocortin-1, a Urocortin-2, a Urocortin-3, a BrainNatriuretic Peptide, a Prostacyclin Synthase, a Growth Hormone, anInsulin-like Growth Factor-1 protein); it can include regions precedingand following the coding region (leader and trailer) as well as, whereapplicable, intervening sequences (introns) between individual codingsegments (exons). “Operably linked” can refer to a functionalrelationship between two or more nucleic acid (e.g., DNA) segments. Inalternative aspects, it can refer to the functional relationship oftranscriptional regulatory sequence to a transcribed sequence. Forexample, a promoter can be operably linked to a coding sequence, such asa nucleic acid used to practice this invention, if it stimulates ormodulates the transcription of the coding sequence in an appropriatehost cell or other expression system. In alternative aspects, promotertranscriptional regulatory sequences can be operably linked to atranscribed sequence where they can be physically contiguous to thetranscribed sequence, i.e., they can be cis-acting. In alternativeaspects, transcriptional regulatory sequences, such as enhancers, neednot be physically contiguous or located in close proximity to the codingsequences whose transcription they enhance.

In alternative aspects, the invention comprises use of “expressioncassettes” comprising a nucleotide sequences used to practice thisinvention, which can be capable of affecting expression of the nucleicacid, e.g., a structural gene or a transcript (e.g., encoding aparacrine protein) in a host compatible with such sequences. Expressioncassettes can include at least a promoter operably linked with thepolypeptide coding sequence or inhibitory sequence; and, in one aspect,with other sequences, e.g., transcription termination signals.Additional factors necessary or helpful in effecting expression may alsobe used, e.g., enhancers.

In alternative aspects, expression cassettes used to practice thisinvention also include plasmids, expression vectors, recombinantviruses, any form of recombinant “naked DNA” vector, and the like. Inalternative aspects, a “vector” used to practice this invention cancomprise a nucleic acid that can infect, transfect, transiently orpermanently transduce a cell. In alternative aspects, a vector used topractice this invention can be a naked nucleic acid, or a nucleic acidcomplexed with protein or lipid. In alternative aspects, vectors used topractice this invention can comprise viral or bacterial nucleic acidsand/or proteins, and/or membranes (e.g., a cell membrane, a viral lipidenvelope, etc.). In alternative aspects, vectors used to practice thisinvention can include, but are not limited to replicons (e.g., RNAreplicons, bacteriophages) to which fragments of DNA may be attached andbecome replicated. Vectors thus include, but are not limited to RNA,autonomous self-replicating circular or linear DNA or RNA (e.g.,plasmids, viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879),and can include both the expression and non-expression plasmids. Inalternative aspects, the vector used to practice this invention can bestably replicated by the cells during mitosis as an autonomousstructure, or can be incorporated within the host's genome.

In alternative aspects, “promoters” used to practice this inventioninclude all sequences capable of driving transcription of a codingsequence in a cell, e.g., a mammalian cell such as a heart, lung,muscle, nerve or brain cell. Thus, promoters used in the constructs ofthe invention include cis-acting transcriptional control elements andregulatory sequences that are involved in regulating or modulating thetiming and/or rate of transcription of a gene. For example, a promoterused to practice this invention can be a cis-acting transcriptionalcontrol element, including an enhancer, a promoter, a transcriptionterminator, an origin of replication, a chromosomal integrationsequence, 5′ and 3′ untranslated regions, or an intronic sequence, whichare involved in transcriptional regulation. These cis-acting sequencestypically interact with proteins or other biomolecules to carry out(turn on/off, regulate, modulate, etc.) transcription.

In alternative embodiments, “constitutive” promoters used to practicethis invention can be those that drive expression continuously undermost environmental conditions and states of development or celldifferentiation. In alternative embodiments, “Inducible” or“regulatable” promoters used to practice this invention can directexpression of the nucleic acid of the invention under the influence ofenvironmental conditions, administered chemical agents, or developmentalconditions.

Gene Therapy and Gene Delivery Vehicles

In alternative embodiments, methods of the invention comprise use ofnucleic acid (e.g., gene or polypeptide encoding nucleic acid) deliverysystems to deliver a payload of a paracrine-encoding nucleic acid orgene, or a paracrine polypeptide-expressing nucleic acid, transcript ormessage, to a cell or cells in vitro, ex vivo, or in vivo, e.g., as genetherapy delivery vehicles.

In alternative embodiments, expression vehicle, vector, recombinantvirus, or equivalents used to practice methods of the invention are orcomprise: an adeno-associated virus (AAV), a lentiviral vector or anadenovirus vector; an AAV serotype AAV5, AAV6, AAV8 or AAV9; arhesus-derived AAV, or the rhesus-derived AAV AAVrh.10hCLN2; anorgan-tropic AAV, or a cardiotropic AAV, or a cardiotropic AAVM41mutant; and/or an AAV capsid mutant or AAV hybrid serotype. Inalternative embodiments, the AAV is engineered to increase efficiency intargeting a specific cell type that is non-permissive to a wild type(wt) AAV and/or to improve efficacy in infecting only a cell type ofinterest. In alternative embodiments, the hybrid AAV is retargeted orengineered as a hybrid serotype by one or more modificationscomprising: 1) a transcapsidation, 2) adsorption of a bi-specificantibody to a capsid surface, 3) engineering a mosaic capsid, and/or 4)engineering a chimeric capsid. It is well known in the art how toengineer an adeno-associated virus (AAV) capsid in order to increaseefficiency in targeting specific cell types that are non-permissive towild type (wt) viruses and to improve efficacy in infecting only thecell type of interest; see e.g., Wu et al., Mol. Ther. 2006 September;14(3):316-27. Epub 2006 Jul. 7; Choi, et al., Curr. Gene Ther. 2005June; 5(3):299-310.

Use of any AAV serotype is considered within the scope of the presentinvention. In some embodiments, a rAAV vector is a vector derived froman AAV serotype, including without limitation, AAV ITRs are AAV1, AAV2,AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10,AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, ormouse AAV ITRs or the like. In some embodiments, the nucleic acid in theAAV comprises an ITR of AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12,AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV or the like.

For example, the rhesus-derived AAV AAVrh.10hCLN2 or equivalents thereofcan be used, wherein the rhesus-derived AAV may not be inhibited by anypre-existing immunity in a human; see e.g., Sondhi, et al., Hum GeneTher. Methods. 2012 October; 23(5):324-35, Epub 2012 Nov. 6; Sondhi, etal., Hum Gene Ther. Methods. 2012 Oct. 17; teaching that directadministration of AAVrh.10hCLN2 to the CNS of rats and non-humanprimates at doses scalable to humans has an acceptable safety profileand mediates significant payload expression in the CNS.

Also, for example, AAV vectors specifically designed for cardiac genetransfer (a cardiotropic AAV) can be used, e.g., the AAVM41 mutanthaving improved transduction efficiency and specificity in themyocardium, see, e.g., Yang, et al. Virol J. 2013 Feb. 11; 10(1):50.

Because adeno-associated viruses (AAVs) are common infective agents ofprimates, and as such, healthy primates carry a large pool ofAAV-specific neutralizing antibodies (NAbs) which inhibit AAV-mediatedgene transfer therapeutic strategies, the methods of the inventioncomprise screening of patient candidates for AAV-specific NAbs prior totreatment, especially with the frequently used AAV8 capsid component, tofacilitate individualized treatment design and enhance therapeuticefficacy; see, e.g., Sun, et al., J. Immunol. Methods. 2013 Jan. 31;387(1-2):114-20, Epub 2012 Oct. 11.

Kits and Instructions

The invention provides kits comprising compositions and methods of theinvention, including instructions for use thereof. As such, kits, cells,expression vehicles (e.g., recombinant viruses, vectors) and the likecan also be provided.

For example, in alternative embodiments, the invention provides kitscomprising compositions used to practice this invention, e.g.,comprising a urocortin-2 (UCn-2) peptide or polypeptide; or aparacrine-encoding nucleic acid, (b) a liquid or aqueous formulation ofthe invention, or (c) the vesicle, liposome, nanoparticle or nanolipidparticle of the invention. In one aspect, the kit further comprisinginstructions for practicing any methods of the invention, e.g., in vitroor ex vivo methods for increasing a desired paracrine level in thebloodstream, or for protecting a cell, e.g., a cardiac or lung cell; orfor treating, preventing or ameliorating diabetes or pre-diabetes.

Formulations

In alternative embodiments, the invention provides compositions andmethods for use in increasing paracrine levels in vivo. In alternativeembodiments, these compositions comprise paracrine-encoding nucleicacids formulated for these purposes, e.g., expression vehicles orparacrine-encoding nucleic acids formulated in a buffer, in a salinesolution, in a powder, an emulsion, in a vesicle, in a liposome, in ananoparticle, in a nanolipoparticle and the like.

In alternative embodiments, the invention provides methods comprisingadministration of urocortin-2 (UCn-2) peptides or polypeptides, orUCn-2-encoding nucleic acids, to treat, ameliorate or prevent a diabetes(including Type 1 and Type 2, or adult onset diabetes) or pre-diabetes,or obesity or excess weight; or to stimulate weight loss, or to act asan appetite suppressant. Accordingly, the invention provides theappropriate formulations and dosages of urocortin-2 (UCn-2) peptides orpolypeptides, or UCn-2-encoding nucleic acids, for same.

In alternative embodiments, the compositions (including formulations ofurocortin-2 (UCn-2) peptides or polypeptides, or paracrine-encoding(e.g., UCn-2-encoding) nucleic acids, can be formulated in any way andcan be applied in a variety of concentrations and forms depending on thedesired in vitro, in vivo or ex vivo conditions, including a desired invivo or ex vivo method of administration and the like. Details ontechniques for in vitro, in vivo or ex vivo formulations andadministrations are well described in the scientific and patentliterature.

Formulations and/or carriers of the paracrine-encoding nucleic acids, orurocortin-2 (UCn-2) peptides or polypeptides, used to practice thisinvention are well known in the art. Formulations and/or carriers usedto practice this invention can be in forms such as tablets, pills,powders, capsules, liquids, gels, syrups, slurries, suspensions, etc.,suitable for in vivo or ex vivo applications.

In alternative embodiments, paracrine-encoding nucleic acids, orurocortin-2 (UCn-2) peptides or polypeptides, used to practice thisinvention can be in admixture with an aqueous and/or buffer solution oras an aqueous and/or buffered suspension, e.g., including a suspendingagent, such as sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia, and dispersing or wetting agents such as anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethylene oxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensationproduct of ethylene oxide with a partial ester derived from fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate).The aqueous suspension can also contain one or more preservatives suchas ethyl or n-propyl p-hydroxybenzoate. Formulations can be adjusted forosmolarity, e.g., by use of an appropriate buffer.

In practicing this invention, the compounds (e.g., formulations) of theinvention can comprise a solution of paracrine-encoding nucleic acids orgenes, or urocortin-2 (UCn-2) peptides or polypeptides, dissolved in apharmaceutically acceptable carrier, e.g., acceptable vehicles andsolvents that can be employed include water and Ringer's solution, anisotonic sodium chloride. In addition, sterile fixed oils can beemployed as a solvent or suspending medium. For this purpose any fixedoil can be employed including synthetic mono- or diglycerides, or fattyacids such as oleic acid. In one embodiment, solutions and formulationsused to practice the invention are sterile and can be manufactured to begenerally free of undesirable matter. In one embodiment, these solutionsand formulations are sterilized by conventional, well knownsterilization techniques.

The solutions and formulations used to practice the invention cancomprise auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents, e.g., sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate and the like. The concentration ofactive agent (e.g., paracrine-encoding nucleic acids or genes) in theseformulations can vary widely, and can be selected primarily based onfluid volumes, viscosities and the like, in accordance with theparticular mode of in vivo or ex vivo administration selected and thedesired results, e.g., increasing in vivo paracrine expression.

The solutions and formulations used to practice the invention can belyophilized; for example, the invention provides a stable lyophilizedformulation comprising paracrine-encoding nucleic acids or genes, orurocortin-2 (UCn-2) peptides or polypeptides. In one aspect, thisformulation is made by lyophilizing a solution comprising aparacrine-encoding nucleic acid or gene, or urocortin-2 (UCn-2) peptidesor polypeptides, and a bulking agent, e.g., mannitol, trehalose,raffinose, and sucrose or mixtures thereof. A process for preparing astable lyophilized formulation can include lyophilizing a solution about2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and asodium citrate buffer having a pH greater than 5.5 but less than 6.5.See, e.g., U.S. patent app. no. 20040028670.

The compositions and formulations of the invention can be delivered bythe use of liposomes (see also discussion, below). By using liposomes,particularly where the liposome surface carries ligands specific fortarget cells, or are otherwise preferentially directed to a specifictissue or organ type, one can focus the delivery of the active agentinto a target cells in an in vivo or ex vivo application.

Nanoparticles, Nanolipoparticles and Liposomes

The invention also provides nanoparticles, nanolipoparticles, vesiclesand liposomal membranes comprising compounds (e.g., paracrine-encodingnucleic acids or genes, or urocortin-2 (UCn-2) peptides or polypeptides)used to practice the methods of this invention, e.g., to deliverparacrine-encoding nucleic acids or genes, or urocortin-2 (UCn-2)peptides or polypeptides, to an individual, a patient or mammalian cellsin vivo or ex vivo. In alternative embodiments, these compositions aredesigned to target specific molecules, including biologic molecules,such as polypeptides, including cell surface polypeptides, e.g., fortargeting a desired cell type, e.g., a mammalian cardiac cell, a kidneycell, a lung cell, a nerve cell and the like.

The invention provides multilayered liposomes comprising compounds usedto practice this invention, e.g., as described in Park, et al., U.S.Pat. Pub. No. 20070082042. The multilayered liposomes can be preparedusing a mixture of oil-phase components comprising squalane, sterols,ceramides, neutral lipids or oils, fatty acids and lecithins, to about200 to 5000 nm in particle size, e.g., to entrap a paracrine-encodingnucleic acid or gene.

Liposomes can be made using any method, e.g., as described in Park, etal., U.S. Pat. Pub. No. 20070042031, including method of producing aliposome by encapsulating an active agent (e.g., paracrine-encodingnucleic acids or genes, or urocortin-2 (UCn-2) peptides orpolypeptides), the method comprising providing an aqueous solution in afirst reservoir; providing an organic lipid solution in a secondreservoir, and then mixing the aqueous solution with the organic lipidsolution in a first mixing region to produce a liposome solution, wherethe organic lipid solution mixes with the aqueous solution tosubstantially instantaneously produce a liposome encapsulating theactive agent; and immediately then mixing the liposome solution with abuffer solution to produce a diluted liposome solution.

In one embodiment, liposome compositions used to practice this inventioncomprise a substituted ammonium and/or polyanions, e.g., for targetingdelivery of a compound (e.g., paracrine-encoding nucleic acids or genes)used to practice this invention to a desired cell type, as describede.g., in U.S. Pat. Pub. No. 20070110798.

The invention also provides nanoparticles comprising compounds (e.g.,paracrine-encoding nucleic acids or genes, or urocortin-2 (UCn-2)peptides or polypeptides) used to practice this invention in the form ofactive agent-containing nanoparticles (e.g., a secondary nanoparticle),as described, e.g., in U.S. Pat. Pub. No. 20070077286. In oneembodiment, the invention provides nanoparticles comprising afat-soluble active agent of this invention or a fat-solubilizedwater-soluble active agent to act with a bivalent or trivalent metalsalt.

In one embodiment, solid lipid suspensions can be used to formulate andto deliver paracrine-encoding nucleic acids or genes, or urocortin-2(UCn-2) peptides or polypeptides, used to practice the invention to apatient, an individual, or mammalian cell in vivo or ex vivo, asdescribed, e.g., in U.S. Pat. Pub. No. 20050136121.

Delivery Vehicles

In alternative embodiments, any delivery vehicle can be used to practicethe methods or compositions of this invention, e.g., to deliverparacrine-encoding nucleic acids or genes, or urocortin-2 (UCn-2)peptides or polypeptides, to practice the methods of the invention invivo or ex vivo. For example, delivery vehicles comprising polycations,cationic polymers and/or cationic peptides, such as polyethyleneiminederivatives, can be used e.g. as described, e.g., in U.S. Pat. Pub. No.20060083737.

In one embodiment, a dried polypeptide-surfactant complex is used toformulate a composition of the invention, wherein a surfactant isassociated with a nucleic acid via a non-covalent bond e.g. asdescribed, e.g., in U.S. Pat. Pub. No. 20040151766.

In one embodiment, a nucleic acid or polypeptide used to practice thisinvention can be applied to cells as polymeric hydrogels orwater-soluble copolymers, e.g., as described in U.S. Pat. No. 7,413,739;for example, a nucleic acid or protein can be polymerized through areaction between a strong nucleophile and a conjugated unsaturated bondor a conjugated unsaturated group, by nucleophilic addition, whereineach precursor component comprises at least two strong nucleophiles orat least two conjugated unsaturated bonds or conjugated unsaturatedgroups.

In one embodiment, a nucleic acid or protein is applied to cells usingvehicles with cell membrane-permeant peptide conjugates, e.g., asdescribed in U.S. Pat. Nos. 7,306,783; 6,589,503. In one aspect, thenucleic acid itself is conjugated to a cell membrane-permeant peptide.In one embodiment, a nucleic acid, protein, and/or the delivery vehicleare conjugated to a transport-mediating peptide, e.g., as described inU.S. Pat. No. 5,846,743, describing transport-mediating peptides thatare highly basic and bind to poly-phosphoinositides.

In one embodiment, electro-permeabilization is used as a primary oradjunctive means to deliver a paracrine-encoding nucleic acids or genesto a cell, e.g., using any electroporation system as described e.g. inU.S. Pat. Nos. 7,109,034; 6,261,815; 5,874,268.

Products of Manufacture, Implants and Artificial Organs

The invention also provides products of manufacture comprising cells ofthe invention (e.g., cells modified to express paracrine proteins, orurocortin-2 (UCn-2) peptides or polypeptides, to practice the methods ofthe invention), and use of cells made by methods of this invention,including for example implants and artificial organs, bioreactorsystems, cell culture systems, plates, dishes, tubes, bottles and flaskscomprising cells modified to express paracrine proteins to practice themethods of the invention. Any implant, artificial organ, bioreactorsystems, cell culture system, cell culture plate, dish (e.g., petridish), cell culture tube and/or cell culture flask (e.g., a rollerbottle) can be used to practice this invention.

In alternative embodiments the invention provides a bioreactor, implant,stent, artificial organ or similar device comprising cells modified toexpress paracrine proteins to practice the methods of the invention; forexample, including implants as described in U.S. Pat. Nos. 7,388,042;7,381,418; 7,379,765; 7,361,332; 7,351,423; 6,886,568; 5,270,192; andU.S. Pat. App. Pub. Nos. 20040127987; 20080119909 (describing auricularimplants); 20080118549 (describing ocular implants); 20080020015(describing a bioactive wound dressing); 20070254005 (describing heartvalve bio-prostheses, vascular grafts, meniscus implants); 20070059335;20060128015 (describing liver implants).

Implanting Cells In Vivo

In alternative embodiments, the methods of the invention also compriseimplanting or engrafting cells, e.g., cardiac, lung or kidney cells,comprising or expressing paracrine-encoding nucleic acids or genes, orurocortin-2 (UCn-2) peptides or polypeptides, used to practice theinvention; and in one aspect, methods of the invention compriseimplanting or engrafting the paracrine-encoding nucleic acids or genes(or cells expressing them), or urocortin-2 (UCn-2) peptides orpolypeptides, in a vessel, tissue or organ ex vivo or in vivo, orimplanting or engrafting the re-programmed differentiated cell in anindividual in need thereof.

Cells can be removed from an individual, treated using the compositionsand/or methods of this invention, and reinserted (e.g., injected orengrafted) into a tissue, organ or into the individual, using any knowntechnique or protocol. For example, de-differentiated re-programmedcells, or re-programmed differentiated cells, can be re-implanted (e.g.,injected or engrafted) using microspheres e.g., as described in U.S.Pat. No. 7,442,389; e.g., in one aspect, the cell carrier comprises abulking agent comprising round and smooth polymethylmethacrylatemicroparticles preloaded within a mixing and delivery system and anautologous carrier comprising these cells. In another embodiment, thecells are readministered to a tissue, an organ and/or an individual inneed thereof in a biocompatible crosslinked matrix, as described e.g.,in U.S. Pat. App. Pub. No. 20050027070.

In another embodiment, the cells of the invention (e.g., cells made bypracticing the methods of this invention) are readministered (e.g.,injected or engrafted) to a tissue, an organ and/or an individual inneed thereof within, or protected by, a biocompatible, nonimmunogeniccoating, e.g., as on the surface of a synthetic implant, e.g., asdescribed in U.S. Pat. No. 6,969,400, describing e.g., a protocol wherea cAMP-incompetent AC can be conjugated to a polyethylene glycol thathas been modified to contain multiple nucleophilic groups, such asprimary amino or thiol group.

In one embodiment, the cells of the invention (e.g., cells made bypracticing the methods of this invention) are readministered (e.g.,injected or engrafted) to a tissue, an organ and/or an individual inneed thereof using grafting methods as described e.g. by U.S. Pat. Nos.7,442,390; 5,733,542.

Any method for delivering polypeptides, nucleic acids and/or cells to atissue or organ (e.g., a lung, kidney, heart) can be used, and theseprotocols are well known in the art, e.g., as described in U.S. Pat. No.7,514,401, describing e.g., using intracoronary (IC), intravenous (IV),and/or local delivery (myocardial injection) of polypeptides, nucleicacids and/or cells to a heart in situ. For example, in alternativeembodiments, aerosol drug particles into the lungs and into thebloodstream, gene therapy, continuous infusions, repeated injectionsand/or sustained release polymers can be used for deliveringpolypeptides, nucleic acids and/or cells to a tissue or organ (e.g., alung, kidney, heart). In alternative embodiments, nucleic acids and/orcells can be given through a catheter into the coronary arteries or bydirect injection into the left atrium or ventricular myocardium via alimited thoracotomy; or delivered into the myocardium via a catheterpassed during cardiac catheterization; or delivered into the pericardialspace.

In alternative embodiments, nucleic acids or proteins used to practicethis invention, or a vector comprising a nucleic acid used to practicethe invention (e.g., an AAV, or adenoviral gene therapy vector), orvesicle, liposome, nanoparticle or nanolipid particle (NLP) of theinvention, and the like, to a tissue or organ (e.g., a lung, kidney,heart); e.g. as described in U.S. Pat. No. 7,501,486, e.g., polypeptidesof the invention comprising an amino acid sequence CRPPR (SEQ ID NO:1),the amino acid sequence CARPAR (SEQ ID NO:2) or a peptidomimeticthereof, or amino acid sequence CPKRPR (SEQ ID NO:3) or a peptidomimeticthereof.

Compositions used to practice this invention can be used in combinationwith other therapeutic agents, e.g. angiogenic agents, anti-thromboticagents, anti-inflammatory agents, immunosuppressive agents,anti-arrhythmic agents, tumor necrosis factor inhibitors, endothelininhibitors, angiotensin-converting enzyme inhibitors, calciumantagonists, antibiotic agents, antiviral agents and viral vectors.

Compositions used to practice this invention can be used forameliorating or treating any of a variety of cardiopathies andcardiovascular diseases, e.g., cardiopathies and cardiovasculardiseases, e.g., coronary artery disease (CAD); atherosclerosis;thrombosis; restenosis; vasculitis including autoimmune and viralvasculitis such as polyarteritis nodosa, Churg-Strass syndrome,Takayasu's arteritis, Kawasaki Disease and Rickettsial vasculitis;atherosclerotic aneurisms; myocardial hypertrophy; congenital heartdiseases (CHD); ischemic heart disease and anginas; acquiredvalvular/endocardial diseases; primary myocardial diseases includingmyocarditis; arrhythmias; and transplant rejections; metabolicmyocardial diseases and myocardiomyopathies such as congestive,hypertrophic and restrictive cardiomyopathies, and/or heart transplants.In alternative embodiments, compositions used to practice thisinvention, e.g., urocortin-2 (UCn-2) peptides or polypeptides, are usedfor treating, ameliorating or protecting (preventing) diabetes orpre-diabetes in a patient or an individual; or suppressing weight gain,or suppressing the appetite, or stimulating or initiating weight loss,in a patient or an individual; or treating, ameliorating or protecting(preventing) diabetes in a patient or an individual.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

EXAMPLES Example 1: Intravenous Delivery of AAV9 Encoding Urocortin-2Increases Cardiac Function in Normal Mice

This example demonstrates the effectiveness of an exemplary embodimentof the invention: intravenous delivery of AAV9/urocortin-2 (orAAV9/UCn2) provided sustained increases in serum UCn2 and LV contractilefunction, indicating the effectiveness of this exemplary embodiment ofthe invention for the treatment of heart failure.

In this study, we developed and tested the relative efficacy of twoadeno-associated virus (AAV) serotypes (AAV5 and AAV9) encodingurocortin-2 (UCn-2), which is a vasoactive peptide in thecorticotropin-releasing factor family that has protean beneficialeffects in animals and patients with heart failure. AAV5.Ucn-2 andAAV9.Ucn-2 (5×10¹¹ genome copies, gc) were delivered by intravenousinjection (IV). Four weeks (wks) after gene transfer, AAV DNA (qPCR) waselevated in liver (AAV5.UCn2: 2,601,839 copies/μg; AAV9.UCn2: 30,121,663copies/μg) and heart (AAV5: 87,635gc/μg; AAV9: 300,529 copies/μg; andmRNA was similarly elevated compared to endogenous UCn2 (AAV5.Ucn-2:68±xx-fold; AAV9.Ucn-2: 8,575).

Left ventricular samples showed Ucn2 mRNA elevation only with AAV9.UCn2,which was increased 28 fold over endogenous mRNA. Plasma Ucn-2 wasincreased (AAV5.UCn2: from 2.7 ng/ml pre to 3.6 ng/ml, p<0.0001;AAV9.UCn2. Finally, associated with increased serum UCn2 levels wereincreases in LV contractile function.

Example 2: Gene Transfer for the Treatment of Cardiovascular Diseases

This example demonstrates the effectiveness of an exemplary embodimentof the invention in obtaining high yield transgene expression in theheart in a manner that can be easily and safely applied.

In alternative embodiments, the invention provides methods usingexpression vehicles, e.g., vectors, encoding a paracrine-type transgene.In this embodiment, the transgene acts as a hormone, having cardiaceffects after being released to the circulation from a distant site. Inalternative embodiments this approach can circumvent the problem ofattaining high yield cardiac gene transfer and enable patients to betreated by a systemic injection during an office visit.

We examined multiple AAV serotype vectors and delivery methods, andsuccessfully completed proof-of-concept studies of paracrine genetransfer. Rats with severe dilated CHF underwent skeletal muscledelivery of an adeno-associated virus 5 (AAV5) vector encodingInsulin-like Growth Factor I (IGFI) under tetracycline regulation. Thisenabled activation of IGFI expression upon adding doxycycline in therat's water supply. The system provided sustained elevation of serumlevels of IGFI and improved function of the failing heart.

In alternative embodiments, a) IGFI gene transfer is used to increasecontractile function; b) AAV vectors and promoters are used forintravenous delivery to provide maximal transgene expression withminimal off-target effects; c) regulated transgene expression is used toenable fine-tuning of serum transgene levels, and allow turningexpression off and on as needed; d) gene transfer ofparacrine-expressing genes, e.g., in a rat model of CHF is used; and e)effective doses of AAV are used, and activators of transgene expressionare used following intravenous delivery of the vector, e.g., in normalpigs, using serum paracrine (e.g., IGFI) as an end-point.

In alternative embodiments, IV injection of an AAV vector with regulatedexpression of selective peptides will, through paracrine-mediatedactions, have favorable effects on the failing heart.

Vector Selection.

In alternative embodiments adeno-associated virus (AAV) vectors areused, enabling long-term transgene expression superior to adenovirus,while avoiding the potential for insertional mutagenesis associated withlentivirus vectors. Persistent serum elevation of Factor IX,erythropoietin and αl-antitrypsin, have been documented in dogs andnonhuman primates, years after single injections of AAV vectors' and wehave confirmed persistent (>1 year) serum elevation of IGFI afterintramuscular injection of AAV5.IGFI-tet in rats in our laboratory.⁵Although recent clinical trials have found that some AAV serotypesincite immune responses,^(6,7) newer generation AAV vectors do notappear to have similar problems in preclinical studies in primates.

AAV Serotypes: In alternative embodiments an AAV serotype AAV2 is used,but in some embodiments, “pseudotyped” AAV vectors are preferred. TheseAAV serotypes, which include AAV5, AAV6, AAV8 and AAV9, are hybridconstructs that include the capsid of AAV2 and unique replicationcomponents that confer their specific nomenclature. In alternativeembodiments, Intravenous delivery of AAV6, AAV8 and AAV9 is used; theseshow substantial distribution and transgene expression in heart, liver,skeletal muscle, and elsewhere.

We found intravenous better than intramuscular AAV5 in increasing serumlevels of IGFI, as illustrate in FIG. 7, which graphically show data offree IGFI serum levels 3 months after IV vs. IM AAV5.IGFI.tet genetransfer: Intravenous delivery in mice (n=3, each group) provided a2-fold increase in serum IGFI after activation of IGFI expression withdoxycycline (On); Intramuscular delivery in rats (n=9 each group)provided a >1.3-fold increase in serum IGFI 5 weeks after activation ofIGFI expression. P values above bars: within group comparison (t-test, 2tails). Change in serum IGFI was greater after intravenous delivery ofAAV5.IGFI.tet (p<0.001).

When given intravenously, AAV9 was superior to AAV5 in terms oftransgene expression in liver and heart, as illustrate in FIG. 8graphically, and by image, illustrates data showing the relativeefficacy of intravenous delivery of exemplary AAV5 and AAV9 constructsof the invention using copy number and transgene expression in liver andheart as endpoints, as illustrated in FIG. 8.

In alternative embodiments, AAV8, like AAV9, provides generalizedexpression, but provides a higher proportion in liver than other organs,a property that, in combination with a liver-specific promoter, wepropose to exploit.

In alternative embodiments, self-complementary AAV vectors (scAAV)vectors are used; they can provide higher transgene expression thantheir single stranded (ssAAV) analogs.⁸ Transgene expression using thessAAV vectors (insert capacity 4.7 kb) is delayed 4-6 w until thecomplementary DNA strand is synthesized. By encoding for thecomplementary DNA strand within the vector, scAAV (insert capacity 3.3kb), enables transgene expression in 2 w and results in higher transgeneexpression vs its ssAAV analog.⁸

Only one regulated expression vector (AAV8.TBG.IGFI.tet) may be amenableto scAAV construction, the others are too large, as illustrated in FIG.10. However, if this vector is selected for the pig studies, ssAAV canbe used to provide better yield for manufacturing the large amountsrequired. The scAAV analog can be used for human use, taking advantageof superior expression, enabling reduced dose requirements, andimproving safety in the clinical trials.

Promoter Vs Target Tissue.

In alternative embodiments, the promoter selected for transgeneexpression in AAV vectors has some tissue-dependence. In alternativeembodiments, promoters used to practice the invention include: chickenβ-actin (CBA); thyroid hormone-binding globulin (TBG, liver-specific);and Rous Sarcoma Virus (RSV) promoters. In this regard, CMV hasconsistently been shown to be a superior promoter in skeletal andcardiac muscle. Recent studies indicate that the CMV promoter issusceptible to methylation in liver, which eventually shuts offtransgene expression. Losing liver expression would reduce serum levelsof transgene—we therefore have elected not to use the CMV promoter,selecting instead similarly robust promoters less susceptible tomethylation: chicken β-actin (CBA); thyroid hormone-binding globulin(TBG, liver-specific); and Rous Sarcoma Virus (RSV) promoters, asillustrated in FIG. 10.

TABLE 2 Tetracycline vs Rapamycin Regulation Feature TetracyclineRapamycin Activator Doxycycline AP22594 Basal Expression (“leak”) Verylow/none None Linear Dose-Response Yes Yes Activator Side-effects Low(avoid Immunosuppressant in pregnancy) Bacterial/Viral Proteins Yes NoUsed in Clinical Trials Not yet Not yet TG, transgene; AP22594, oralrapamycin analog, 100-fold less immune suppression vs rapamycin¹⁴

Regulated Expression. In alternative embodiments, using long-termexpression conferred by AAV-mediated gene transfer, transgene expressionis regulated to turn off expression if unexpected untoward effects areseen. Regulated expression would also enable intermittent rather thanconstant delivery. In alternative embodiments, the system can beconfigured so that the activator either turns off or turns on transgeneexpression. In instances where nearly constant transgene expression isrequired, an “Off” system is desirable (e.g., one takes the oralactivator only when transgene expression is not desired, for example inthe event of adverse effects). In alternative embodiments, in instanceswhere intermittent transgene expression is required, an “On” system isdesirable (e.g., one takes the oral activator only for those times thattransgene expression is desired).

These alternative embodiments enable tight control, and, when tailoredfor the specific disease treated, provide a means to take the leastamount of activator. In alternative embodiments, regulated expressionsystems are used, e.g., ecdysone, tamoxifen, tetracycline,rapamycin⁹⁻¹²; the large size of the ecdysone system may require atwo-vector strategy that would be difficult to develop for clinical genetransfer because of regulatory constraints. The tamoxifen system, whilenot as cumbersome, requires a less tolerated activator than thetetracycline system (tamoxifen vs doxycycline). In alternativeembodiments, only two of the available options (tetracycline andrapamycin regulation) may be suitable, and these are the only systemsthat have been tested in large animal models.^(3,4) Both of thesesystems possess analogous features (Table 2, above): the gene ofinterest is controlled by an engineered transcription factor inducibleby an activating drug (tetracycline or a rapamycin analog).

Tetracycline-Regulated Expression. In alternative embodiments theinvention uses tetracycline-regulated expression in the setting of genetransfer:

a) Basal expression of transgene (“leak”). Newer rtTA variants, such asthe one we propose and have used in recent studies (rtTA2^(S)-M2),provide robust tetracycline-dependent expression with no basalactivity,¹³ unlike previous rtTA constructs.

b) Chronic use of tetracycline vis-à-vis patient tolerability and offtarget effects.

-   -   The tet-regulation system has been extensively studied;¹¹ in        vitro studies show that doxycycline-stimulated transgene        expression begins at 0.001 ng/ml and reaches a maximum at 0.1        μg/ml, a 10-fold reduction in EC₅₀ vs the first generation        system.¹³ In humans, a single oral dose of 200 mg doxycycline        provides mean plasma and tissue concentrations of 1.5 μg/m at 24        h,¹⁴ 15-fold higher than required for maximal expression. A        single daily dose of doxycycline of 10-20 mg may suffice for        complete activation of transgene expression in human subjects.¹⁵        Doses of 200 mg/d are well-tolerated by patients using oral        doxycycline chronically for acne and chronic infections.^(14,16)    -   ORACEA® (doxycycline 40 mg orally once daily) is approved by the        FDA for continuous use to treat rosacea.¹⁶ This dose, 80% lower        than the 200 mg dose required to treat infection, provides        anti-inflammatory effects that treat rosacea, but does not have        anti-microbial effects, and does not lead to the development of        antibiotic-resistant organisms (11 years of clinical data). Each        capsule contains 40 mg of anhydrous doxycycline as 30 mg of        immediate-release and 10 mg of delayed-release beads. Subjects        with allergies to tetracycline, increased photosensitivity,        pregnant or lactating women, or children less than 9 years old        (discoloration of teeth, possible reduced long bone growth)        should not use doxycycline. In 5 years of clinical use, the most        common side effect was mild gastrointestinal complaints.¹⁶    -   Tetracyclines may attenuate matrix metalloproteinase expression        and activity, and have an impact on left ventricular (LV)        remodeling when administered in the first few days after        myocardial infarction (MI).¹⁷ However, in the proposed        preclinical studies, doxycycline is administered 5 weeks after        MI, when LV chamber dilation and scar formation are stable and        equal among groups. We have previously documented that        doxycycline does not influence LV remodeling, TIMP, or MMP        expression in the proposed murine MI-induced CHF model.¹⁸ In the        clinical setting, tetracycline will not be used in the acute        phase of MI.

c) Immune responses to the components of the rtTA system. Immuneresponses to the tet-regulator were not identified in a long-term studythat used AAV4.tet and AAV5.tet gene transfer (intra-retinal) innon-human primates,^(3,15) where they saw sustainedtetracycline-dependent transgene expression for the 2.5 year duration ofthe study. We do not see inflammation in mouse hearts expressing highlevels of rtTA,^(18,19) or in mice and rats after AAV5-mediatedregulated expression of IGFI using the rtTA2^(S)-M2 regulation element.⁵It appears that intramuscular delivery of AAV.tet in nonhuman primates,unlike intra-retinal or vascular delivery, does lead to attenuation ofregulated expression, owing to immune responses to the bacterial andvirus component of the transactivator fusion protein.²⁰ Immune responseto the tet-regulator can be and simultaneously, the rapamycin-regulationsystem, which does not possess bacterial or virus proteins, and is notassociated with provocation of the immune response, can be determined.⁷See Table 2 for strengths and limitations of tet- and rapamycinregulation.

Rapamycin-Regulated Expression. In alternative embodiments, a macrolidesirolimus (rapamycin), a product of the bacterium Streptomyceshygroscopicus, is used: it was initially developed as an anti-fungalagent, but was found to have anti-proliferative and immunosuppressanteffects. Currently it is used clinically: a) to prevent rejection inorgan transplantation (2 mg P.O. (per os, orally), once daily, whichprovides mean serum levels of 12±6 ng/ml); and b) in drug-eluting stentsto reduce restenosis after angioplasty, owing to its antiproliferativeeffects. Rapamycin increases life span in mice, appears to forestalldeleterious effects of aging,²¹ and is used as an adjuvant in thetreatment of glioblastoma multiforme.²² Rapamycin binds cytosolicFK-binding protein 12 (FKBP12) and inhibits the mammalian target ofrapamycin (mTOR) signaling pathway. A serine/threonine protein kinase,mTOR influences cell growth and proliferation, and promotes cellsurvival. Rapamycin's usefulness vis-à-vis gene therapy lies in itsdimerization properties, a feature that is exploited in therapamycin-regulated expression system. In this system, the DNA-bindingand activation domains of an engineered transcription factor areexpressed separately as fusion proteins, which are cross linked, andthereby activated, by the addition of a bivalent “dimerizing” drug, inthis case rapamycin or a rapamycin analog.¹² Expression isdose-dependent, reversible, and is triggered by nanomolar concentrationsof activator.¹² The rapamycin system contains no virus or bacterialproteins, and is therefore unlikely to incite an immune response. Inmacaques, intramuscular injection of AAV1 encoding erythropoietinprovided up to 6 year (yr) Rap-regulated expression (26 separateinduction cycles) with no decline in levels of erythropoietin, and noimmune response to the regulation elements.⁴ Immunosuppression is apotential disadvantage to rapamycin. However, this problem can becircumvented by using an oral rapamycin analog (AP22594), whichactivates transgene expression as effectively as rapamycin, exhibitsminimal immune suppression, and does not inhibit mTOR.⁴ Furthermore, asan activator, weekly rather than daily doses are effective, furtherreducing off-target effects. The dose-response relationship of orallyadministered AP22594, and its maximal dose-intervals, starting with oraldoses used effectively in macaques (0.45 mg, once weekly), can bedetermined in pigs.⁴

Insulin-Like Growth Factor I (IGFI)

Selection of IGFI. Growth hormone (GH) exerts many of its effectsthrough activation of IGFI. IGFI exerts many of its effects through Akt.Because of the convergence of signaling from GH through IGFI to Akt, theselection of IGFI over GH or Akt must be defended. Increased GHexpression would be predicted to increase serum glucose and bloodpressure—deleterious effects that are avoided by selecting IGFI.Increased expression of Akt would be expected to reduce apoptosis, buthave other potentially favorable effects not provided by Akt, such asincreased angiogenesis. We therefore selected IGFI gene transfer for ourinitial preclinical CHF studies, and recently shown that IGFI genetransfer improves function of the failing rat heart⁵ (see FIGS. 1-8 andTables 4 & 5).

IGFI Signaling. IGFs, initially known as somatomedins, are a family ofpeptides that mediate many of the anabolic and mitogenic activities ofGH. Two somatomedins with structural and metabolic similarities toinsulin were isolated from human plasma in 1978 and named IGFI andIGFII. IGFI (somatomedin C) subsequently was shown to be the IGFregulated by circulating GH. IGFI has 70 amino acids in a single chainwith 3 disulfide bridges and a molecular weight of 7.6 kD. Initiallythought to be generated only by the liver, it has been shown to beproduced by many tissues, including intestine, brain, kidney, lung andheart. Liver-specific deletion of the IGFI gene in rats does not alternormal growth and development,²³ indicating that IGFI, expressed widelyin other tissues including heart, regulates growth and developmentthrough local tissue release in a paracrine manner.

IGFI belongs to a family of proteins including ligands (IGFI, IGFII,insulin), six known binding proteins (IGFBP 1-6), and cell surfacereceptors including IGFI and insulin receptors.²⁴ IGFI is translated asa pre-pro peptide which includes an amino terminal signal peptide, A, B,C and D domains and a variable carboxyl terminal E peptide. There arethree known isoforms of pro-IGFI in humans (pro-IGFIa, pro-IGFIb andpro-IGFIc) that differ only in the amino acid composition of thevariable E peptide. IGF binding proteins (IGFBP) act as carrier proteinsand prolong the half-life of IGF by inhibiting degradation.²⁴ Almost all(98%) of IGFI circulates bound predominantly (80%) to IGFBP-3.²⁴

IGFI and IGFII display high affinity binding to the IGFI receptor in alltissues except liver. The IGF receptor shares 60% homology with theinsulin receptor and contains a tyrosine kinase domain. Receptor bindingof IGFI results in autophosphorylation of tyrosine residues. Thisactivates the receptor, producing phosphorylation of substratesincluding the insulin receptor substrate, which activates multiplesignaling cascades including the PI3 kinase/Akt and mitogen activatedprotein kinase (MAPK) pathways, and others, many of which havebeneficial cardiovascular effects (see below Sections and Table 3).

Effects of Increased IGFI. Increased serum IGFI lowers serum insulinlevels, increases insulin sensitivity and improves lipid profiles.²⁴However, infusion of IGFI protein can cause hypotension andhypoglycemia.²⁵ GH, which opposes insulin activity, increases serumglucose levels. The ability of IGFI to increase glucose uptake in theheart may play a role in post ischemic recovery of LV function afterIGFI administration. IGFI increases muscle blood flow and hasvasodilator activity, through receptor dependent and independent effectsand nitric oxide production.²⁶ The combined metabolic and vasodilatoreffects of high-dose intravenous IGFI infusion in humans may causelightheadedness and flushing—lower doses increase cardiac performance,do not affect blood pressure or serum glucose, and are unassociated withsymptoms.^(25,27)

IGFI receptor activation is responsible for numerous cellular responsesincluding regulation of gene expression, stimulation of myogenesis, cellcycle progression, immune modulation, and steroidogenesis. In the heart,IGFI and the IGFI receptor/PI3K/Akt signaling pathway have beneficialeffects on cardiac myocyte function, growth and survival. Moreover, IGFexhibits angiogenic effects,²⁸ increases cardiac contractile function innormal^(25,29,30) and failing hearts,^(27,29,30-33) and inhibitsapoptosis^(34,35,38). These features make IGFI attractive for CHFtherapy (Table 3).

TABLE 3 IGFI: Beneficial Cardiovascular Effects Feature MechanismSpecies Ref ↓ SVR Vasodilation via NO R, P, H 25, 26, 32, 39* ↑ LVInotrope; vasodilation R, D, H 25, 29, 30, 36 ↑ LV Function in CHFInotrope ↑Ca²⁺ R, D, P 27, 29-33, 39* handling ↑ Cardiac Protection ↓Apoptosis via Akt M, R 34, 35, 38 ↑ LV Mass CM prolif; M, R 31, 36, 45,46 ↓ Apoptosis via Akt ↑ Blood flow Angiogenesis R 28 SVR, systemicvascular resistance; CO, cardiac output; EF, ejection fraction; LV, leftventricular; prolif, proliferation; M, mouse; R, rat; D, dog; P, pig; H,human; 39* used in GH, which elevated IGFI 2-fold

IGFI Protein in Treatment of Heart Disease (Table 3)

Preclinical Studies. The effects of administering recombinant human IGFIor GH protein in animal models of heart diseases have been studied. IGFIis a positive inotrope in isolated rat hearts and ferret papillarymuscle; GH has no inotropic effect in the same tissues.²⁹ Similarinotropic effects of IGFI were found in isolated papillary muscles fromdogs with pacing induced heart failure.³⁰ IGFI administered to normalrats for four weeks increased cardiac function and resulted inconcentric LV hypertrophy.³¹ IGFI and GH administered together for twoweeks was associated with increased LV dP/dt and LV hypertrophy innormal rats.³⁶ Administration of IGFI prior to myocardial ischemia andreperfusion in rats decreased creatine kinase release and reducedapoptosis.³⁴ Combined IGFI and GH³² or IGFI alone³³ administered fourweeks after MI increased LV function in rats. GH given to rats for fourweeks after MI increased LV systolic function,³⁷ reduced cardiacfibrosis, cardiac myocyte apoptosis, and increased survival.³⁸ In thepacing model of CHF in pigs, GH increased serum IGFI, increased LVfunction and reduced LV wall stress.³⁹

Clinical Studies. Clinical use of GH or IGFI protein has receivedconsiderable attention, although there is a paucity of largeplacebo-controlled studies. The acute hemodynamic effects of IGFIinfusion were studied in a blinded placebo-controlled crossover study ofCHF patients (n=8). Four-hour infusions of IGFI increased cardiacoutput, decreased vascular resistance, and reduced right atrial andwedge pressures.²⁷ Chronic administration of IGFI protein has not beenevaluated in patients with CHF. Use of GH protein in patients with CHFhas produced equivocal results. Two small uncontrolled and unblindedstudies in a total of 14 patients with CHF reported that three months ofGH protein therapy increased serum IGFI, LV function and clinicalstatus.^(40,41) Randomized placebo-controlled trials of GH (protein)given for up to 3 months in patients with CHF did not alter LV functionor clinical status.^(42,43) The most recent literature review of GHprotein therapy concludes that evidence for efficacy in ischemic andidiopathic clinical CHF is lacking, perhaps due to the kinetics ofpeptide administration.⁴⁴ Thus, in alternative embodiments, the genetransfer methods of this invention, by providing sustained IGFIexpression, can be superior to IGFI protein therapy.

Increased Expression of Cardiac IGFI or GH. Cardiac-directed expressionof human IGFI in rats, with its attendant increase in cardiac myocyteIGFI production, nearly doubles serum IGFI levels. These rats haveincreased heart weights with cardiac myocyte hyperplasia, but noincrease in cardiac myocyte volume.^(35,45) After MI, reduced cardiacmyocyte apoptosis, and increased phosphorylation of Akt were found.³⁵Cardiac-directed expression of IGFI attenuates age-related cellsenescence with reductions in telomerase activity, telomere shorteningand DNA damage. These rats show increased Akt activation, and increasedLV function at 22 months of age vs age-matched transgene negativelittermates.⁴⁶ Co-expression of cardiac IGFI in a cardiomyopathicbackground (crossbreeding paradigm) appears to prevent cardiacapoptosis, LV remodeling and LV dysfunction.⁴⁷ However, since CHF wasnever present, this strategy is not equivalent to treating alreadyexisting CHF, an approach that is a central theme in the currentproposal.

To determine if GH gene transfer would influence LV remodeling after MI,rat cardiac muscle was directly injected with adenovirus encoding GH(Ad.GH) at the time of coronary occlusion.⁴⁸ Injections were made in theborder zone between jeopardized and viable myocardium. Six weeks afterMI and gene transfer, favorable effects were seen on LV end-diastolicdimension, LV dP/dt and wall thickness in the infarct region. The samescientists subsequently showed that Ad.GH injected into the infarctborder zone of rats three weeks after coronary artery occlusionincreased LV dP/dt and attenuated LV dilation and wall thinning threeweeks after injection.⁴⁹ GH gene transfer during or 3 w after MIappeared to have beneficial effects on LV remodeling.

When adenovirus encoding IGFI (Ad.IGFI) was injected into thejeopardized perfusion bed just before coronary occlusion in rats, theextent of infarction was reduced 50%, an effect thought primarily to bethe result of reduced apoptosis.⁵⁰ This study did not address theeffects of IGFI gene transfer on LV remodeling after MI. Adenovirusmediated gene transfer of IGFI has been shown to reduce hypoxia-inducedmyocyte apoptosis in vitro, and, in a rat ischemia reperfusion model,prior injection of adenovirus encoding IGFI reduced infarct sizeapproximately 50% (p<0.003), although the transgene was expressed inonly about 15% of the ischemic region, consistent with a regionalparacrine effect. The effect of expressing IGFI in the globally failingheart has not been explored.

Potential IGFI Adverse Effects

Survival. Disruption of the GH/IGFI system appears to increase, notdecrease longevity in rats with normal cardiac function.⁵¹ However, wepropose to increase IGFI expression in the setting of severe CHF, whichportends markedly increased short-term mortality. No data suggest thatIGF inhibition increases longevity in CHF. To the contrary, increasedserum IGFI in humans reduces the incidence of CHF and mortality.^(52,53)Epidemiological studies have shown that people with low serum IGFI areat increased risk of developing ischemic heart disease. In theFramingham study, individuals above the median value for serum IGFI hada 50% reduced incidence of CHF compared to those below themedian.^(52,53) A recent report shows that angiotensin converting enzymeinhibitors (ACEI), which prolong life in CHF, increases IGFIsignaling.⁵⁴ Our data show that IGFI gene transfer increases function ofthe failing rat heart, and we propose to determine whether there also isa survival benefit.

Cancer. Clinical epidemiological studies report a correlation betweenincreases in serum IGFI levels (>2-fold elevations) and prostate andpremenopausal breast cancer,⁵⁵ but there is no indication that thiscorrelation is causal. It is noteworthy that the incidence of prostatecancer increases with age, while serum IGFI concentration decreases.⁵⁵In cancer patients, increased serum IGFI may originate in the tumor.Indeed, increased expression of IGFI in prostate epithelium of ratselevates serum IGFI concentrations and can lead to prostate neoplasia.⁵⁶Increased serum IGFI concentrations may also result from changes innutritional status in cancer patients. One could speculate that IGFI mayincrease tumor growth through angiogenesis and reduced apoptosis.Cardiac-directed expression of IGFIb, with attendant sustainedelevations in serum IGFI, is not associated with prostate or breastcancer and combined increases in serum IGFI and GH do not increase theincidence of prostate, breast or lung cancer in patients withacromegaly.⁵⁴ The role of IGFI in the genesis or progression of canceris theoretical. It seems prudent that therapies that increase IGFIexpression should limit serum concentrations of IGFI, and also provide ameans to stop expression if desired. We propose to achieve these goalsby using gene transfer of a regulated expression vector, which increasesIGFI concentrations in the serum and thereby has beneficialcardiovascular effects.

Novelty of Studies. These studies are focused on the development of IGFIgene transfer for clinical CHF. IGFI (or GH) gene transfer has not beenused in clinical CHF. No double-blinded placebo-controlled clinicaltrial of GH/IGFI protein in CHF has been successful, perhaps due to therelatively short biological half-life of GH/IGFI protein, a problem thatwould be overcome by gene transfer. Although GH and IGFI cardiac genetransfer have been used prior to coronary occlusion to reduce infarctsize in animal studies, no previous study has examined IGFI genetransfer for CHF per se. In addition, the proposed paracrine approachusing systemic delivery of a long term and regulated expression vectoris new, and can be applied to other paracrine-based peptides to treat avariety of cardiovascular diseases.

Summary Because of the limitations of preclinical and clinical studiesvis-à-vis predictable benefits of peptide administration of IGFI in thetreatment of severe CHF, and the theoretical promise of paracrine-basedgene transfer of IGFI, we embarked on studies in our laboratory (seePreliminary Data), designed to circumvent impediments and shortcomingsof continuous or chronic intermittent intravenous peptide infusion.

Other Beneficial Peptides.

Although the use of IGFI is compelling, it should be emphasized that theparacrine gene therapy methods of the invention are also suited for anycirculating peptide with beneficial cardiovascular effects. For example,urocortin-2 is a recently discovered vasoactive peptide in thecorticotropin-releasing factor family that acts viacorticotropin-releasing factor type 2 receptors, which are robustlyexpressed in the heart and vasculature. Infusions of urocortin-2 peptidehave protean beneficial effects in animals and patients with heartfailure.⁵⁷ BNP is another biologically effective peptide for thetreatment of clinical CHF that could be delivered in a similar manner.Moreover, in pulmonary hypertension, prostacyclin analogs can beeffective in treating pulmonary hypertension, but current agents(epoprostenol and trepostinil) require constant systemic injection, andthe treatment itself is associated with high morbidity.⁵⁸ In alternativeembodiments, methods of the invention provide a regulated expressionvector encoding prostacyclin synthase as a paracrine-type gene therapyof pulmonary hypertension. Indeed, any current peptide therapeutic thatrequires prolonged or chronic intermittent intravenous infusion, wouldlend itself to this hormone-like gene transfer approach.

AAV & Immune Response in Clinical Studies. Long-term transgeneexpression after intramuscular or intravascular delivery of AAV vectorshas been the rule rather than the exception in rodents. However, studiesin patients have been bedeviled by limited expression due to immuneresponses to the transgene and, at times, the AAV vector per se.⁶ Twoconclusions emerge from these and other studies. 1) Intramuscular (ascompared to intravascular) AAV delivery generally provokes increasedimmune response to the transgene and AAV capsid; and 2) success inrodents, due to their relative immune tolerance, does not always predictsuccess in humans. Rodent and pig studies can be designed with humans inmind:

-   -   AAV serotypes (AAV8 and AAV9) can be selected that are least        likely to be associated with pre-existing neutralizing        antibodies in human subjects.⁵⁹ For example, AAV8 is associated        with the lowest prevalence of anti-AAV neutralizing antibodies        (19% vs 59% for AAV1 and 50% for AAV2). Moreover, among the        minority of human subjects with AAV8/9 antibodies, 75-90% of        those subjects possess low titers, making AAV8 and AAV9 the        current optimal choices vis-à-vis anticipated immune response.⁵⁹        Human sera possesses almost no seropositivity to rhesus-derived        AAV vectors, such as AAVrh.32.33,⁶⁰ providing an alternative        vector if AAV8 and AAV9 prove unsuitable, although preclinical        and clinical experience with AAVrh.32.33 is limited.    -   Intramuscular injection of AAV vectors can be avoided because        they may incite immune responses in larger animals.⁶    -   Two species-specific IGFI proteins can be used: rat and pig.        Both rat and porcine IGFI can be used. The use of        species-specific IGFI will reduce immune responses to the        transgene. Clinical trials can be performed with the optimal        vector encoding human IGFI.

Intravenous delivery of AAV8 and AAV9 is appealing because of itssimplicity, and because it is likely to achieve the highest serum levelsof therapeutic transgene at the lowest possible AAV dose. Althoughseroprevalence to these AAV vectors is important in pigs and primates,including humans, it has not been an important factor in rodents.Preliminary sampling of pigs from our vendor show no evidence of AAV8 orAAV9 antibodies in 7 of the 9 pigs tested.

In alternative embodiments, expression of a transgene of the inventionis limited to a single organ, e.g, if such a strategy providestherapeutic serum levels of that transgene. For example, an exemplaryvector of the invention is AAV8 with a hepatocyte-specific promoter(TBG, human thyroid hormone-binding globulin).

Paracrine-Based Gene Transfer Using IGFI.

Although we selected IGFI for these proof-of-concept studies, inalternative embodiments, the invention comprises use of any of thecandidate genes outlined herein, and any of these genes would beeffective for the intended effect. For example, the invention providesmethods and compositions that effectively deliver any paracrinepolypeptide, e.g., a mammalian cardiotonic peptide, a Serelaxin, aRelaxin-2, a Urocortin-2, a Urocortin-1, a Urocortin-3, a BrainNatriuretic Peptide, a Prostacyclin Synthase, a Growth Hormone, anInsulin-like Growth Factor-1, or any combination thereof; or, a humanUrocortin-2, a Urocortin-1, a Urocortin-3, a Brain Natriuretic Peptide,a Prostacyclin Synthase, a Growth Hormone, an Insulin-like GrowthFactor-1, or any combination thereof.

We engineered an exemplary AAV5 vector encoding rat IGFI (type A) thatis under control of a tetracycline response element (TRE): FIG. 1illustrates an exemplary construct of the invention comprising AAV5encoding IGF1; this exemplary AAV5 vector provides regulated expressionof IGFI: ITR, inverted terminal repeat; TRE, tetracycline responseelement; IGFIAU1, Insulin-like Growth Factor-I; SVpA, polyA from SV40viral genome (bidirectional); rtTA2^(S)M2, reverse tetracyclinecontrolled transactivator; CMV, human cytomegalovirus early genepromoter. Total insert size, 2823 bp, fits into a scAAV5 vector(capacity 3.3 kb).

The coding sequence includes a signal peptide to ensure extracellularsecretion of IGFI. We have used this vector (AAV5.IGFI.tet) in genetransfer experiments in cultured cardiac myocytes: FIG. 2A illustratesdata from studies where cultured neonatal rat cardiac myocytes wereinfected with AAV5.IGFI.tet (10,000 gc/cell, 2 d); the gels illustratedshow that IGFI expression was induced by doxycycline (+Dox) (2 μg/ml, 3d), but did not occur in the absence of doxycycline (−Dox). IGFI wasdetected in media by anti-AU1 antibody by immunoblotting. FIG. 2Billustrates data where in the same experiments cardiac myocytes werelysed in Akt lysis buffer (10 min, 4° C.) and centrifuged (12,000×g, 10min); total Akt and phospho-Akt were detected by anti-Akt andanti-phospho-T308-Akt antibodies. IGFI expression was associated withAkt activation. After infection, transgene expression was undetectable(no “leak”) until activation with doxycycline (FIG. 2A).

Our vector (FIG. 1) contains a more recent rtTA variant (rtTA2^(S)-M2),which provides robust dox-dependent expression and low or absent basalactivity, unlike previous rtTA constructs.¹³

Regulated IGFI Expression in Cultured Cardiac Myocytes

Cultured neonatal rat cardiac myocytes underwent gene transfer withAAV5.IGFI-tet (10⁴ gc/cell, 2 days). As graphically illustrated in FIG.3, subsequently, doxycyline (2 μg/ml) was added to media, and IGFI mRNAexpression was quantified using real-time RT-PCR. Expression of IGFImRNA was increased (versus (vs) unstimulated) by 1.5-fold within 30 min,and reached a peak of 14-fold elevation by 24 hrs. At 48 hrs, IGFI mRNAwas somewhat less (10-fold), reflecting doxycycline degradation. Toturn-off IGFI expression doxycycline was removed using four sequentialPBS washes (“off-wash,” see FIG. 3). IGFI mRNA rapidly decreased afterdoxycycline withdrawal.

Skeletal Muscle Delivery of AAV5.IGFI.Tet Improves Function of theFailing Heart

Skeletal Muscle Gene Transfer. We initially performed studies in murineheart failure after indirect intracoronary delivery of AAV5.IGFI.tet(FIG. 1), finding substantial improvements in function of the failingheart after cardiac-targeted delivery. However, proof-of-concept studiesto demonstrate the efficacy of a paracrine-based transfer, would requireskeletal muscle delivery of the vector. For these pivotal studies, weused intramuscular delivery of AAV5.IGFI.tet in the tibialis anteriormuscle of rats.⁵ AAV5 was selected because of its well-known highexpression levels after IM injection in skeletal muscle. In allinstances we have found IGFI expression in media (cell cultureexperiments), and long term IGFI expression in heart (murine CHF model)and in serum (rat model after IM injection, mouse after IV injection),and corresponding improvement in function of failing heart.⁵

In the rat study, we first examined the feasibility of skeletal muscleinjection of AAV5.EGFP to provide long-term transgene expression, asillustrated in FIG. 4A: illustrating photomicrographs showing EGFPexpression in unilateral tibialis anterior muscle 3 weeks afterAAV5.EGFP gene transfer in rats. Contralateral uninjected tibialisanterior muscle from the same animal shows no expression of EGFP. FIG.4B is Table 4, which summarizes data from the echocardiography measuringthe effects of Skeletal Muscle IGFI Expression in CHF.

MI Model of CHF & Experimental Protocol

MI was induced in rats by proximal left coronary occlusion, resulting inlarge transmural infarction and severe impairment of LV function. Oneweek after MI, rats with impaired LV function received 2×10¹² genomecopies (gc) of AAV5.IGFI.tet in the anterior tibialis muscle. Four weekslater (5 w after MI), rats with LV ejection fraction (EF)<35% wererandomly assigned to two groups: one group received doxycycline indrinking water to activate IGFI expression (IGF-On; n=10) and the otherdid not receive doxycycline (IGF-Off; n=9). Ten weeks after MI (5 wafter activation of IGFI expression), LV size and function were assessedby echocardiography and hemodynamic studies; FIG. 5 illustrates theexperimental protocol for AAV5.IGFI.tet skeletal muscle gene transfer inCHF.

Outcome. IGF-On rats showed increased LV ejection fraction (p=0.02) andreduced LV end-systolic dimension (p=0.03) (Table 4, see FIG. 4B).Furthermore, LV contractile function, assessed by the rate of pressuredevelopment (LV+dP/dt) during dobutamine infusion, was increased afterinitiation of IGFI expression (p=0.001) (Table 5, see FIG. 26). Inaddition, favorable changes in cardiac output (p=0.007) and stroke work(p=0.003) were observed (Table 5). Serum IGFI was increased 5 wk aftertransgene activation (IGF-Off: 164±24 ng/ml; IGF-On: 218±11 ng/ml;p=0.008; n=9 each group). These data indicate that skeletal muscleinjection of AAV5.IGFI.tet enables tetracycline-activated expression,increases serum IGFI levels, and improves function of the failingheart.⁵ In alternative embodiments, less immunogenic AAV vectors can beused, and they can be used intravenously rather than in an intramuscularinjection to circumvent inciting immune responses, and test tworegulated expression systems.

Cardiac Apoptosis and Fibrosis (FIG. 6)

FIG. 6 illustrates the effects of AAV5.IGFI-tet gene transfer on cardiacapoptosis and fibrosis. FIG. 6A graphically illustrates data from TUNELstaining that indicated that activation of IGFI expression (IGF-On) wasassociated with reduced cardiac myocyte apoptosis (p<0.0001; 2-wayANOVA), which was reduced more in the border than remote region. FIG. 6Billustrates picrosirius red-stained sections of the uninfarctedintraventricular septum from IGF-Off and IGF-On rats that showed reducedcardiac fibrosis, and collagen fractional area was reduced (p=0.048);FIG. 6C graphically illustrates this data from the IGF-Off and IGF-Onrats.

Intravenous vs Intramuscular Delivery of AAV5.IGFI.tet. In preliminarystudies, we determined whether intravenous gene transfer could increasecirculating IGFI levels. One week after intravenous delivery ofAAV5.IGFI.tet (5×10¹⁰ gc per mouse, tail vein) mice were randomlyassigned to one of two groups: one group received doxycycline indrinking water to activate IGFI expression (IGF-On) and the other didnot receive doxycycline (IGF-Off). Since the majority of circulatingIGFI is bound to IGFI binding proteins (IGFBPs) with high affinity andis biologically inactive, we measured free serum IGFI, the bioactiveIGFI form, which was 2-fold higher in IGF-On than in IGF-Off mice 3months after activation of IGFI expression (FIG. 7, next page). Usingthe intramuscular AAV5.IGFI.tet (2×10¹² gc per rat) gene transferstrategy outlined in Section 2.2.1.2., we found a 1.3-fold increase offree serum IGFI in IGF-On group than IGF-Off group 5 weeks afteractivation of IGFI expression (FIG. 7). These data suggest thatintravenous delivery of AAV5.IGFI.tet is more effective thanintramuscular delivery vis-à-vis serum IGFI concentrations.

Moreover, an intravenous strategy is likely to circumvent provocation ofimmune response, which has been observed following intramusculardelivery of AAV.⁶ These experiments provide pivotal feasibility data forour studies.

Intravenous Delivery: AAV5 vs AAV9.

We next determined the relative efficacy of intravenous delivery of AAV5vs AAV9, using copy number and transgene expression in liver and heartas endpoints, as illustrated in FIG. 8. We used self-complementary (sc)AAV vectors, enabling earlier expression vs single-strand (ss) AAVvectors. Mice received intravenous scAAV5.CMV.EGFP or scAAV9.CMV.EGFP(5×10¹¹gc) and were killed 21 d later. PCR primers directed to commonsequences in both vectors were used to compare AAV DNA copy number inliver and heart. In liver, AAV9 (vs AAV5) provided 3-fold increases inboth AAV DNA copies and in EGFP expression; in heart, a 5-fold increasein AAV DNA copies and an 8-fold increase in EGFP expression were seen.These data show that, compared to intravenous AAV5, AAV9 can providehigher serum levels of transgene.

Methods

FIG. 10 illustrates exemplary vectors and vector designs of theinvention: Using intravenous delivery of three vectors selected frompreliminary studies and biological features, the relative merits ofwidely distributed and expressed AAV8 and AAV9 (FIG. A), and AAV8 with aliver-specific promoter (FIG. 10B) can be determined. The criterion foreffectiveness can be serum levels of IGFI 6 weeks (w) after delivery. Anoptimal AAV vector is used to generate two regulated expression vectors(Tet and Rap), which can be compared following intravenous delivery inrats, as illustrated in FIG. 10 C-F. The criterion for effectiveness canbe serum levels of IGFI, this time examined 16 w after activation oftransgene expression (20 w after delivery).

FIGS. 10A & B. AAV vectors for the initial studies in rats to determinethe best AAV serotype for subsequent studies. These vectors encode ratIGFI (unregulated), driven by CBA (AAV8 & AAV9) or TBG (AAV8). The bestof these, based on serum IGFI levels and duration of expression, can beused to undergo subsequent studies to determine the optimal regulationsystem.

FIG. 10C-F. Candidate vectors for studies in rats to determine theoptimal regulated expression system. Using the best AAV vector from theinitial studies (above), 2 regulated expression vectors are generatedand tested: one with Tetracycline-regulation, the other withRapamycin-regulation. These vectors encode regulated expression of ratIGFI, driven by RSV (AAV8 & AAV9) or TBG (AAV8). The CBA promoter is toolarge for the Rapamycin-regulation vector, so RSV is used instead. Thebetter of these two regulation systems is selected for generation of theoptimal vector for the subsequent studies in normal pigs, encodes forregulated expression of porcine IGFI. ITR, inverted terminal repeat;TRE, tetracycline response element; IGFI, Insulin-like growth factor-I;SVpA, polyA from SV40 virus genome (bidirectional); rtTA2^(S)M2, reversetetracycline controlled transactivator; SV40en, simian virus 40enhancer; TBG Prom, thyroid hormone-binding globulin promoter; RSV Prom,Rous sarcoma virus promoter; FRB-p6, part of FRAP, a rapamycininteracting protein, combined with a subunit of transcription factorNF-κB (p65); IRE, internal transcription reentry site; ZF, zinc fingerHD1 DNA binding domain; FKBP, FK506 binding protein; pA, minimalpolyadenylation segment; ZBD, zinc finger HD DNA binding domain (8copies).

We do not anticipate that immune responses to AAV will play an importantrole in rats, although such responses are important in dogs, pigs,humans and other primates. Immune responses should be carefullyassessed. AAV biodistribution (e.g., using qPCR using primers amplifyingcommon sequences in all vectors) and toxicity (e.g., using histologicalanalysis) can be quantified.

Group Size. The primary criterion for success can be serum level ofIGFI, which has a coefficient of variation of 20%. To detect a 30%difference in serum IGFI between groups, assuming an α error of 0.05 anda β error of 0.10, will require a group size of n=10.

Example 3: Delivery of AAV8 Encoding Urocortin-2 Increases CardiacFunction

This example demonstrates that in alternative embodiments of methods ofthis invention a paracrine transgene acts as a hormone and has cardiaceffects after being released to the circulation from a distant site.This exemplary approach can circumvent the problem of attaining highyield cardiac gene transfer and enable patients to be treated by asystemic injection during an office visit. Furthermore, this exemplaryapproach can eliminate the need for intravenous (IV) delivery oftherapeutic peptides and thereby circumvent repeated and prolongedhospital stays, high morbidity, and enormous economic costs. Inalternative embodiments, the most suited vector to achieve these goalsis the adeno-associated virus type 8 (AAV8), which provides long termand extensive expression after intravenous delivery in rodents, pigs,and primates.

In alternative embodiments of methods Urocortin-2, a recently discoveredcorticotropin releasing factor family vasoactive peptide, is used as atherapeutic transgene. Urocortin-2 can act via corticotropin-releasingfactor type 2 receptors, which are robustly expressed in the heart andvasculature. Studies in animals and patients with congestive heartfailure have shown favorable hemodynamic effects of urocortin-2 peptideinfusions, including increased contractile function independent ofloading, indicating direct cardiac effects. We established thatintravenous delivery of AAV8 using the chicken β-actin promoter providessustained high serum levels of UCn2 and increases function of thefailing mouse heart.

To select the best specific embodiments to practice this aspect of theinvention, studies in mice and pigs can be carried out, e.g.: a)determine regulated transgene expression to enable fine-tuning of plasmatransgene levels, and allow turning expression off and on as needed; andb) determine the safety, efficacy, and mechanism of action ofurocortin-2 gene transfer, using this exemplary paracrine-based approachin an art-accepted animal model, a mouse model of CHF. Also, use ofnormal pigs can determine: a) the minimally effective vector doserequired to increase serum UCn2; b) biodistribution of the vector andtransgene; and c) toxicity.

Potential advantages of paracrine gene transfer methods of the inventionover IV peptide infusion are shown in Table 1 (above). In alternativeembodiments, practicing methods of the invention allows circumvention ofinfection and reduced repeated and prolonged hospital stays, therebyreducing costs. In alternative embodiments, systemic vector delivery isan advantage in paracrine gene transfer by providing the highest levelof expression for any given AAV dose. The potential safety and efficacyof this approach was recently demonstrated in an early phase genetherapy clinical trial in patients with hemophilia B,² a study that hasrestored hope in gene therapy. In alternative embodiments, paracrinegene transfer methods of the invention can be suited for any circulatingpeptide with beneficial cardiovascular effects.

In alternative embodiments, AAV is used to enable longer transgeneexpression than adenovirus, and avoid insertional mutagenesis associatedwith retrovirus. Persistent transgene expression has been shown in largeanimals years after a single injection of AAV vectors.⁶⁻¹⁰ We haveconfirmed this in mice¹¹ & rats. Although recent clinical trials havefound that some AAV serotypes incite immune responses after IMinjection,^(12,13) newer generation AAV vectors (AAV5, 6, 8 and 9) donot have similar problems in primates.¹⁴ IV AAV delivery is superior toIM vis-à-vis serum transgene levels, and AAV9 and AAV8 are superior toAAV5¹⁵ (and unpublished data). Moreover, pre-existing anti-AAV8antibodies are not as prevalent in humans (19%) as are other AAVserotypes including AAV1 & AAV2 (50-59%).¹⁶ Our data, graphicallyillustrated in FIG. 11, indicate that IV AAV8 is the optimal vector anddelivery route to attain sustained increased levels of serum UCn2 for aparacrine approach. FIG. 11 illustrates data from: IV delivery ofAAV9.CMV.UCn2 (9.CMV), AAV9.CBA.UCn2 (9.CBA) vs AAV8.CBA.UCn2 (8.CBA);where the data indicated that all vectors were associated withsubstantial increases in serum UCn2 6 w later. Numbers in bars denotesample size for each group; p value from ANOVA. ITR, inverted terminalrepeat; SVpA, polyA from SV40 viral genome; UCn2, urocortin-2; CBA,chicken β-actin promoter; CMV enhancer, human cytomegalovirus enhancer.

Despite its robustness in striated muscle, the CMV promoter issusceptible to methylation and inactivation in liver,¹⁷ and our dataindicate that promoters less susceptible to methylation are superior.Indeed, although CMV provided a sustained 2-fold increase in UCn2 afterIV vector delivery, use of the chicken β-actin (CBA) promoter resultedin 15.7-fold increase in serum UCn2, as illustrated in FIG. 11. Thehepatocyte-specific thyroid hormone-binding globulin (TBG) promoter alsocan be used.

In alternative embodiments, the constructs and methods of the inventionallow for regulated expression, e.g., turning off expression. Because ofthe potential for long-term expression conferred by AAV gene transfer,the ability to turn off expression is desirable in the event thatuntoward effects develop. Regulated expression also enables theflexibility of intermittent rather than constant transgene delivery. Inalternative embodiments, the constructs and methods of the invention useregulated expression systems such as e.g.: ecdysone, tamoxifen,tetracycline, rapamycin.¹⁸⁻²¹ The size of the ecdysone system requires atwo-vector strategy and tamoxifen presents issues with toxicity.Tetracycline and rapamycin regulation systems (Table 2) have been testedin large animal models^(9,10,22-26)

TABLE 2 Tetracycline vs Rapamycin Regulation Feature TetracyclineRapamycin Activator Doxycycline AP22594 Basal Expression (“leak”) Verylow/none None Linear Dose-Response Yes Yes Activator Side-effects Low(avoid Immunosuppressant in pregnancy) Bacterial/Viral Proteins Yes NoUsed in Clinical Trials Not yet Not yet TG, transgene; AP22594, oralrapamycin analog, 100-fold less immune suppression vs rapamycin¹⁴

In alternative embodiments, the constructs and methods of the inventionuse a tet-regulation system, which has been extensively studied.²⁷Unlike previous rtTA constructs, rtTA variants of this invention (e.g.,rtTA2^(S)-M2), provide robust tet-dependent expression with no basalactivity (i.e. no “leak”)^(11,26,28,29,30) and 10-fold highersensitivity to tetracycline (maximum transgene expression activation at0.1 μg/ml).³⁰ A single daily dose of doxycycline of 10-20 mg may sufficefor complete activation of transgene expression in humansubjects.^(26,31) Doses of 200 mg/d are well tolerated by patients usingoral doxycycline chronically for acne and chronic infections.^(31,32)Tetracyclines may attenuate matrix metalloproteinase (MMP) activity andaffect LV remodeling when administered in the first few days after MI.²⁴We have previously shown that doxycycline does not influence LVremodeling, TIMP, or MMP expression in the proposed murine MI-inducedCHF model, where doxycycline is given 5 w after MI.²⁵ In clinicalsettings, tetracycline will not be used in the acute phase of MI.

Immune responses to components of the rtTA system, a potential problem,were not identified in a study of AAV4.tet and AAV5.tet gene transfer(intraretinal) in non-human primates,⁹ where tetracycline-dependenttransgene expression persisted for the 2.5 year duration of the study.We do not see inflammation in mouse hearts expressing high levels ofrtTA,^(25,28,29) or in mice after AAV5-mediated regulated expression ofIGFI using the rtTA2^(S)-M2 regulation element.¹¹ It appears that IMdelivery of AAV.tet in nonhuman primates, unlike intra-retinal orvascular delivery, does lead to attenuation of regulated expression,owing to immune responses to the bacterial and virus components of thetransactivator fusion protein.³³ Immune response to the tet-regulatorand the rapamycin-regulation system, which does not possess bacterial orvirus proteins and is not associated with provocation of the immuneresponse, can be simultaneously tested.¹⁰ See Table 2 for strengths andlimitations of tet- & rapamycin regulation.

In the rapamycin regulation system, transgene expression is triggered bynanomolar concentrations of rapamycin or a rapamycin analog, which isdose-dependent and reversible.²¹ Rapamycin is used clinically tosuppress immune response, forestalls deleterious effects of aging inmice²³ and inhibits glioblastoma multiforme³⁴ by blocking the mammaliantarget of rapamycin (mTOR) signaling pathway.³⁵ The oral rapamycinanalog AP22594, which activates transgene expression as effectively asrapamycin, exhibits minimal immune suppression, and does not inhibitmTOR.^(10,35-37) Pigs can be used to determine the dose-responserelationship of orally administered AP22594, and its required dosingintervals, starting with oral doses similar to those used effectively inmacaques (0.45 mg/kg, once weekly).¹⁰

In alternative embodiments, the constructs and methods of the inventionexpress in vivo Urocortin-2, including UCn1, UCn2 and UCn3 (38-40 aminoacids (aa)), which belong to the corticotropin-releasing factor (CRF)family. These peptides can stimulate corticotropin-releasing factorreceptors 1 and 2 (CRF1, CRF2). UCn1 binds to CRFR1 & CRFR2, but UCn2 &Ucn3 exclusively bind CRFR2,³⁸⁻⁴¹ which are expressed in cardiacmyocytes, vasculature, gut, brain and skeletal muscle.^(42,43,44)Although UCn1 was found in LPS-induced inflammation and was implicatedin tissue permeability,^(45,46) UCn2's effects, which are diverse, havebeen associated with favorable biological effects, owing in part to itsaffinity for CRFR2. UCn2's effects are not entirely cAMP-dependent. Forexample, CRFR2 desensitization after UCn2 binding induces PI3K/Aktsignaling via translocation of β-arrestin. In addition, increased ERK1/2signaling occurs via disassociation of G protein β & γ subunits.^(47,48)These cAMP-independent events contribute to reduced cardiac myocyteapoptosis. Peptide infusions of UCn2 in preclinical and clinical CHFhave consistently shown favorable effects on LV function, and reducedactivation of the sympathoadrenal axis.⁴⁹⁻⁵¹

As listed in Table 3, below, among many beneficial effects, UCn2infusion using methods and compositions of the invention can increasecontractile function independent of loading conditions, indicatingdirect cardiac effects.⁵² The mechanisms for inotropic effects have notbeen defined. Recent studies suggest beneficial effects on Ca²⁺handling,⁵³ action potential duration,⁵⁴ ischemia-reperfusioninjury,⁵⁵⁻⁵⁷ and the renin-aldosterone system.⁴⁹ The safety and efficacyof UCn2 infusion has been confirmed in large animal models ofCHF,^(58,59) and in normal human subjects & patients with CHF.^(50,51) Arecent editorial promotes its use in Class 3 & 4 CHF.⁶⁰

TABLE 3 Urocortin-2: Beneficial Cardiovascular Effects Feature MechanismSpecies Ref ↓ SVR Vasodilation M, S, H 44, 57-59 via Cr FR 2 ↑ CO & EFInotrope; M, S, H 44, 57-59 vasodilation ↓ Cardiac Work ↓ SVR and LAP M,S, H 44, 57-59 ↑ LV Diastolic Lusotrope M, R, S, H 44, 52, 56 Function ↑Diuresis ↑ RBF & Na S, H 49, 57-59 excretion; ↓ RAS ↑ LV function All ofthe above M, R, S, H 44, 49, 56, 57, 59 in CHF are reported ↓ LV IRInjury & Unknown M, R 53-55 apoptosis SVR, systemic vascular resistance;CRFR2, corticotropin-releasing factor receptor-2; CO, cardiac output;EF, left ventricular ejection fraction; LV, left ventricular; LAP, leftatrial pressure; RBF, renal blood flow; RAS, renin-angiotensin system;CM, myocyte; IR ischemia-reperfusion; M, mouse; R, rat; S, sheep; H,human.

Since plasma half-life of UCn2 is 15 min,⁵¹ chronic infusion isrequired. In contrast, in alternative embodiments, paracrine-based UCn2gene transfer of the invention can circumvent impediments associatedwith chronic peptide infusions, as noted in Table 1, above. Byexpressing only species-specific UCn2 in the two species proposed,immune responses to the transgene will be abrogated.

Paracrine-Based Gene Transfer Proof of Concept.

We proved that paracrine gene transfer via IM injection of AAV5 encodingInsulin-like Growth Factor-I (AAV5.IGFI) improves function of thefailing rat.¹¹ We now also have shown that IV delivery of AAV8 encodingUCn2 not only provides sustained high levels of serum UCn2 (>15-foldincrease), but increases function of normal and failing hearts.

Selection of AAV Vector and Promoter.

It was clear from previous published studies that IV AAV8 or AAV9 wouldprovide higher levels of transgene expression than other AAV serotypes,and CMV or CBA promoters, which generally are the most robust, would beoptimal. Therefore we engineered an AAV8 & two AAV9 vectors encodingmurine UCn2 driven by CMV or CBA to determine which vector would mosteffectively increase serum UCn2, as illustrated in FIG. 11. Acommercially available UCn2-specific ELISA was used. AAV9.CMV raisedserum UCn2 2.3-fold, which, while lower than the other 2 vectors, may besufficient for a therapeutic response. However, AAV8.CBA was associatedwith a 15.7-fold rise in serum UCn2 (AAV8.CBA.UCn2: 109±7 ng/ml, n=9;Control: 7±1 ng/ml). Such a high level of serum UCn2 would enablereducing the AAV8 dose. The superiority of AAV8.CBA and AAV9.CBA overAAV9.CMV may reflect either CBA's relative robustness or CMV'ssusceptibility to methylation and inactivation in liver.¹⁷ We thereforeselected AAV8.CBA for additional studies.

AAV8.CBA.UCn2 Distribution & Expression after Intravenous Delivery.

In alternative embodiments, the constructs and methods of the inventionexpress in vivo by a paracrine-based gene transfer strategy UCn2, andcan be used to increase serum levels of UCn2. Alternative embodiments donot require that UCn2 expression be present in the heart per se, becauseit is the effects of circulating UCn2 and its effects on the heart andvasculature that will provide the therapeutic effects of the transgene,effects that do not require UCn2 expression in cardiac myocytesthemselves.

Liver Expression of UCn2.

The 15.7-fold increase in serum UCn2 documented 6 w after IV delivery ofAAV8.CBA.UCn2 (5×10¹¹ gc; see FIG. 11) was associated with atime-dependent increase in UCn2 mRNA expression in liver, as illustratedin FIG. 12, that plateaued 4-6 weeks after delivery, which correlatedwell with the steady rise in serum UCn2. FIG. 12A graphicallyillustrates a time course of UCn2 mRNA expression in liver afterAAV8.CBA.UCn2 (5×10¹¹ gc, IV). Liver UCn2 expression (each bar is meanvalue from 2 mice) reached a plateau 4-6 weeks after delivery, whichcorrelated with the plateau seen with serum UCn2 (data not shown). FIG.12B graphically illustrates data showing UCn2 mRNA expression in LV 6weeks (w) after AAV8.CBA.UCn2 (5×10¹¹ gc, IV). Similar high levels ofUCn2 mRNA were seen in skeletal muscle samples (data not shown).

Cardiac Expression of UCn2.

Although cardiac expression of UCn2 is not required for the beneficialeffects of the paracrine-based gene therapies of the invention, wedocumented substantial increases in UCn2 mRNA expression in LV samples 6w after IV delivery of AAV8.CBA.UCn2, see FIG. 12B. In alternativeembodiments, a construct of the invention, including e.g., AAV8,including AAV8 DNA presence and UCn2 mRNA, can be delivered to and/orexpressed in any organ or other organs, including skeletal muscle, lung,brain, kidney, spleen, small intestine, bone marrow.

UCn2 Gene Transfer in Normal Mice.

To determine if UCn2 gene transfer increased LV function, we deliveredAAV8.UCn2 (5×10¹¹ gc) or saline (control) by intravenous (IV) deliveryin normal mice. Five weeks after UCn2 gene transfer, mice underwent aninvasive procedure in which Millar catheters (1.4 F) were placed in theLV chamber to measure pressure development. Data acquisition andanalyses were blinded to group identity. UCn2 gene transfer increased LVcontractile function (LV+dP/dt) (FIG. 13A, left); −dP/dt also wasreduced, indicating enhanced LV relaxation (FIG. 13B, right panel). Noadverse effects on LV mass, histology, or LV structure or function weredetected. FIG. 3 graphically illustrates: LV function in normal mice 6weeks after IV delivery of AAV8.CBA.UCn2 (vs saline-injected controlmice. FIG. 3A: LV +dp/dt; FIG. 3B. LV −dP/dt. Values represent mean±SE.Number in bars denotes group size. UCn2 gene transfer increased bothcontractile function and cardiac relaxation.

UCn2 Gene Transfer in Mice with CHF.

We used proximal left coronary occlusion to induce severe CHF in mice, amodel that we have used extensively and that mimics aspects of clinicalischemia-based CHF.²⁵ As shown in the protocol (FIG. 12A), 3 w aftercoronary occlusion, we performed echocardiography to confirm severe LVdysfunction and chamber dilation. We then randomly assigned enrollees toreceive IV delivery of AAV8.CBA.UCn2 (5×10¹¹ gc per mouse) or anequivalent volume of saline. Five weeks after randomization, miceunderwent repeat echocardiography and measurement of LV pressuredevelopment and decay and their first derivative, LV +dP/dt. Dataacquisition and analyses were blinded to group identity. Despite markedLV dysfunction that was present at the time of UCn2 gene transfer, LVfractional area change (FAC %), an ejection fraction surrogate, wasincreased (FIG. 14B). UCn2 gene transfer also increased LV systolic(LV+dP/dt) and diastolic (LV −dP/dt) function (FIG. 14C). Peak LV+dP/dtwas increased to a value that approached normal, confirming that theproposed strategy merits development as a novel therapy for CHF. FIGS.14B and 14C illustrate data showing the effects of UCn2 transfer on thefailing heart: FIG. 14A: 3 w after MI and development of CHF, micereceived IV AAV8.UCn2 or saline; 5 w after gene transfer (8 w after MI),LV function was assessed (blinded studies); FIG. 14B. UCn2 gene transferincreased LV fractional area change (% FAC); FIG. 14C. UCn2 genetransfer increased LV peak +dP/dt and peak −dP/dt, indicating markedbenefits in systolic & diastolic LV function of the failing heart.

UCn2 Gene Transfer:

Effects on Cardiac Ca²⁺ Handling

C2.5.1.UCn2 Gene Transfer Alters Expression of SERCA2a.

AAV8.CBA.UCn2 gene transfer (5×10¹¹ gc, IV) was associated withincreased expression of SERCA2a mRNA and protein in LV samples obtainedfrom mice 4 w after gene transfer (FIG. 15). These changes would beanticipated to promote Ca²⁺ availability to the myofilament, and therebyto increase both systolic and diastolic function, as we have observed innormal and failing hearts following UCn2 gene transfer (FIGS. 13 and14), providing a plausible mechanism by which UCn2 gene transferincreases LV function. Similar effects of UCn2 peptide have beendescribed in isolated cardiac myocytes.⁵³

FIG. 15 illustrates data (FIG. 15A, by graph, FIG. 15B, by immunoblot)where normal mice received IV delivery of AAV8.CBA.UCn2 (5×10¹¹ gc) orsaline (CON); and four weeks later, LV samples from the UCn2 genetransfer group showed a 2-fold increase in SERCA2a protein expression.Immunoblotting signal was normalized to TnI content. Numbers in barsdenote group size. These changes in SERCA2a expression would beanticipated to promote Ca²⁺ availability at the myofilament, and therebyincrease LV systolic and diastolic function.

UCn2 Gene Transfer & Ca²⁺ Transients.

Cardiac myocytes (CM) were isolated from mice 4 w after AAV8.CBA.UCn2(5×10¹¹ gc, IV). Mice that had received IV saline were used as controls.During the measurement, cardiac myocytes from UCn2 mice were incubatedwith 24 nM UCn2 peptide to mimic serum UCn2 levels in vivo. Cardiacmyocytes from mice receiving UCn2 gene transfer showed altered Ca²⁺transients with reduced t½, as illustrated in FIG. 16: Ca²⁺ transientsfollowing UCn2 gene transfer: FIG. 16A graphically illustrates that UCn2gene transfer increased the rate of Ca²⁺ decline; FIG. 16B graphicallyillustrates that time-to-Ca²⁺ transient decay was shortened in cardiacmyocytes from mice that had received UCN2 gene transfer 4 w prior.Experiments were repeated three times. Bars denote mean +SE; numbers inbars denote number of cardiac myocytes; numbers above bars indicate pvalue.

UCn2 is Cardioprotective.

To test UCn2's effects on hypoxic injury, we treated cultured neonatalrat cardiac myocytes with sodium azide (NaN₃), which irreversibly bindsthe heme cofactor in cytochrome oxidase and inhibits mitochondrialrespiration, mimicking hypoxia-induced cytotoxicity. UCn2 treatmentprotected cardiac myocytes from injury as reflected morphologically andby reduced LDH release, as illustrated in FIG. 17. UCn2 also protectsisolated cardiac myocytes from hypoxia-reoxygenation injury (p<0.001;data not shown). FIG. 17 shows data that UCn2 protects cultured neonatalrat cardiac myocytes from hypoxic injury: FIG. 17A illustrates that UCn2(60 nM) preserves morphological normality 24 hr after NaN₃ (10 mM)treatment; FIG. 17B graphically illustrates that UCn2 reduced LDHrelease after NaN₃ treatment (p<0.001).

Effects on CREB and β-Catenin.

LV samples were obtained from mice 4 w after AAV8.CBA.UCn2 (5×10¹¹ gc,IV). Mice that had received IV saline were used as controls. LV samplesfrom mice that had received UCn2 gene transfer showed increasedphosphorylation of CREB (a 3-fold increase, p<0.01, FIG. 18A). CREB is atranscriptional factor that enables CRE-mediated gene expression in theheart. In addition, UCn2 gene transfer was associated with a 2-foldincrease in LV β-catenin phosphorylation (p<0.0001, FIG. 18B). Increasedβ-catenin phosphorylation reduces β-catenin accumulation in theintercalated disks of cardiac myocytes and thereby reduces cardiacstiffness and diastolic dysfunction. This may contribute to ourobservation that UCn2 gene transfer increases LV relaxation in normaland failing hearts. FIG. 18 graphically illustrates that phosphorylationof both CREB (FIG. 18A) and β-catenin (FIG. 18B) was detected in LVsamples 4 w after IV delivery of UCn2.CBA.UCn2. Control mice received IVsaline.

Non-Cardiac Effects of UCn2 Gene Transfer.

IV delivery of AAV8.CBA.UCn2 (5×10¹¹ gc) has a favorable effect onglucose metabolism—an anti-diabetic effect. For example, mice thatreceived UCn2 gene transfer are resistant to hyperglycemia induced byhigh fat diet (HFD), a model of Type 2 diabetes used in preclinicalstudies (FIG. 19A). Reduced glucose levels are due to increased glucoseutilization as seen in glucose tolerance testing of HFD-fed mice (FIG.19B). FIG. 19 illustrates data showing UCn2 affects glucose regulation.Mice received IV delivery of AAV8.CBA.UCn2 (5×10¹¹ gc, n=8) or saline(n=8), & standard chow for 3 w. A small reduction in fasting bloodglucose was seen in the UCn2 group. Mice then received a high fat diet(HFD) for 8 w. Hyperglycemia was seen in Controls, as expected, but UCn2mice maintained normal blood glucose levels. FIG. 19B. Mice received IVdelivery of AAV8.CBA.UCn2 (5×10¹¹ gc, n=8) or saline (n=8) & HFD for 2months & glucose tolerance tests conducted. Fasted mice received glucose(2 mg/g body weight, IP) and glucose levels measured. Results indicatethat UCn2 gene transfer promotes glucose utilization and protectsagainst diet-induced hyperglycemia.

FIG. 20 illustrates exemplary constructs of the invention:Abbreviations: ITR, inverted terminal repeat; TRE, tetracycline responseelement; SVpA, polyA from SV40 viral genome (bidirectional); rtTA2SM2,reverse tetracycline controlled transactivator; SV40en, simian virus 40enhancer; TBG Prom, thyroid hormone-binding globulin promoter; RSV Prom,Rous sarcoma virus promoter; FRB-p6, part of FRAP, a rapamycininteracting protein, combined with a subunit of transcription factorNF-κB (p65); IRE, internal transcription reentry site; ZF, zinc fingerHD1 DNA binding domain; FKBP, FK506 binding protein; pA, minimalpolyadenylation segment; ZBD, zinc finger HD DNA binding domain (8copies).

Example 4: Akita Mouse Model of Type 1 Diabetes Mellitus Responsive toUrocortin 2 Gene Transfer

The Akita Mouse is accepted in the art as a Model for Type 1 DiabetesMellitus. The mouse is characterized by:

-   -   Spontaneous mutation in Ins2 gene, leading to single amino acid        substitution in insulin molecule—a monogenic model of Type 1        Diabetes Mellitus    -   Alters insulin folding, which leads to beta-cell toxicity and        depletion    -   Heterozygotes viable and fertile; homozygotes rarely live 12        weeks    -   Autosomal dominant (easy to breed); male >female phenotype    -   By 4 weeks old: hyperglycemia, polydipsia, polyuria    -   Plasma insulin levels 10-15% of normal mean life span 305 days        (normal 690 days); 80% mortality at 1 year    -   Used to evaluate nephropathy, neuropathy, retinopathy

The rationale for the experiments, to determine whether urocortin-2(UCn2) gene transfer would be effective in an insulin deficient (ratherthan a purely insulin resistant state), was based on data indicatingthat the mechanism by which UCn2 gene transfer is effective in promotingglucose disposal in insulin resistant states does not affect the usualsignaling elements involved in insulin resistance. Instead, it appearedthat UCn2 was operating independently of insulin-signaling and promotingglucose disposal via previously undescribed mechanisms. Since Type 1 DMis not generally associated with striking abnormalities in insulinresistance, testing whether UCn2 gene transfer was effective in theAkita mouse would provide supportive data that UCn2's mechanism ofaction involves an independent pathway for glucose disposal. Inaddition, such a discovery would have significant impact clinically,potentially providing a new therapeutic approach to patients with Type 1diabetes.

Fasting Glucose and Hemoglobin A1c:

Sixteen (16) 3-month old male Akita mice (19 grams body weight) receivedintravenous (IV) delivery of adeno-associated virus type 8 (AAV8, 2×10¹³genome copies/kg. Eight received AAV8.Null (control) and 8 received AAV8encoding murine UCn2, both with CBA promoters. Data were collected 8-10weeks after vector delivery. The results (FIG. 21) show reduced fastingglucose (p<0.0001) in mice that received AAV8.UCn2. Mean fasting glucosewas 270 mg/dL in AAV8.Null mice, but was reduced to 108 mg/dL in micethat received AAV8.UCn2. Sustained improvement in glucose control wasindicated by significant reductions in hemoglobin A1c (p=0.002) (FIG.22).

Glucose Tolerance Test:

The same 16 mice described above underwent glucose tolerance testing toquantify glucose disposal when challenged with a glucose load after a12-hr fast (2.5 g/kg). These data (FIG. 23) indicate a more rapidglucose disposal rate (p=0.01), as indicated by reduced area under theglucose concentration-time curve. These data, which show some overlapbetween the two groups, must be confirmed by measuring insulinsensitively using hyperinsulinemic euglycemic clamps, studies that areunderway.

Insulin Release and Insulin Tolerance Testing.

Because it is plausible that UCn2 may influence insulin release orinsulin sensitivity, we tested insulin release (FIG. 24) and insulintolerance (FIG. 25). Insulin release was assessed after a 12 hr fastfollowed by an oral glucose load (2.5 g/kg). Insulin tolerance wastested after a 4 hr fast and an insulin challenge (0.75 U/kg, IV). Anunexpected finding was that mice that had received UCn2 gene transfer 10weeks prior, showed superior insulin release both 60 min (p=0.003) and120 min (p=0.004) after glucose challenge (FIG. 24). Insulin tolerancetesting indicated a reduced EC50 for glucose disposal in mice that hadreceived UCn2 gene transfer (FIG. 25; p<0.003).

In summary, the data show that UCn2 gene transfer increases glucosedisposal independently of insulin. UCn2 gene transfer increases insulinsensitivity and UCn2 gene transfer may increase glucose-induced insulinrelease. Thus, these data support the use of UCn2 gene transfer for thetreatment of insulin deficient states, such as encountered in clinicalType 1 DM.

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A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method for treating type 1 diabetes in a mammalcomprising: injecting an adeno-associated viral vector (AAV) comprisinga nucleic acid sequence encoding Uracortin 2 (UCN2) operably linked to apromoter to a mammal with type 1 diabetes intravenously (IV) such thatincreased insulin release and decreased hyperglycemia in the mammaloccurs, thereby treating the type 1 diabetes in the mammal.
 2. Themethod of claim 1, wherein the vector is selected from AAV8 or AAV9. 3.The method of claim 1, wherein the promoter is selected from a chickenbeta-actin (CBA), thyroid hormone-binding globulin (TBG), or RousSarcoma Virus (RSV).
 4. The method of claim 1, wherein the mammal is ahuman.
 5. The method of claim 1, wherein the mammal is a canine,non-human primate or feline subject.
 6. The method of claim 1, whereinthe method comprises between about 1 to 5 intravenous (IV) injections.7. The method of claim 1, wherein the vector is AAV-DJ or AAVrh10. 8.The method of claim 1, wherein the adeno-associated viral vector (AAV)is formulated as a buffered aqueous solution.
 9. The method of claim 1,wherein the adeno-associated viral vector (AAV) is formulated in aliposome.
 10. The method of claim 9, wherein the liposome is amultilayered liposome.
 11. The method of claim 1, wherein theadeno-associated viral vector (AAV) is formulated in a nanoparticle ornanolipoparticle.